High density immobilization of nucleic acids

ABSTRACT

Processes and kits for immobilizing a high density of nucleic acids on an insoluble surface, which are particularly useful for mass spectrometric detection of nucleic acids, are disclosed. Arrays containing the immobilized nucleic acids and use of the immobilized nucleic acids in a variety of solid phase nucleic acid chemistry applications, including nucleic acid synthesis (chemical and enzymatic), hybridization and/or extension, and sequencing, are provided. Serial and parallel dispensing tools that can deliver defined volumes of fluid to generate multi-element arrays of sample material on a substrate surface are further provided. Tools provided herein can include an assembly of vesicle elements, or pins, wherein each of the pins can include a narrow interior chamber suitable for holding nanoliter volumes of fluid. Methods for dispensing tools that can be employed to generate multi-element arrays of sample material on a substrate surface are also provided. The tool can dispense a spot of fluid to a substrate surface by spraying the fluid from the pin, contacting the substrate surface or forming a drop that touches against the substrate surface. The tool can form an array of sample material by dispensing sample material in a series of steps, while moving the pin to different locations above the substrate surface to form the sample array, The prepared sample arrays may be passed to a plate assembly that disposes the sample arrays for analysis by mass spectrometry.

RELATED APPLICATIONS

For U.S. National Stage purposes, this application is acontinuation-in-part of a U.S. application filed as Ser. No. 08/947,801on Oct. 8, 1997, to Maryanne J. O'Donnell-Maloney, Charles R. Cantor,Daniel P. Little and Hubert Köster, entitled “Methods of High DensityImmobilization of Nucleic Acids, and Uses Thereof” which is acontinuation-in-part of U.S. application Ser. No. 08/746,055, filed Nov.6, 1996, now abandoned, to Maryanne J. O'Donnell-Maloney, Charles R.Cantor and Hubert Köster, entitled “High Density Immobilization ofNucleic Acid Molecules”. This application is also a continuation-in-partof U.S. application Ser. No. 08/746,055, U.S. application Ser. No.08/786,988, filed Jan. 23, 1997, to Daniel P. Little, Maryanne J.O'Donnell-Maloney, Charles R. Cantor and Hubert Köster, entitled“Systems and Methods for Preparing and Analyzing Low Volume AnalyteArray Elements” and U.S. application Ser. No. 08/787,639, filed Jan. 23,1997 now U.S. Pat. No. 6,024,925, to Daniel P. Little and Hubert Köster,entitled “Systems and Methods for Preparing Low Volume Analyte ArrayElements”. For international purposes, benefit of priority is claimed toeach of these applications.

This application is related to U.S. Pat. Nos. 5,547,835, 5,622,824,5,605,798.

Where permitted the subject matter of each of the above-noted patentapplications and patents is herein incorporated in its entirety.

BACKGROUND OF THE INVENTION

In the fields of molecular biology and biochemistry, as well as in thediagnosis of diseases, nucleic acid hybridization has become a powerfultool for the detection, isolation and analysis of specificoligonucleotide sequences. Typically, such hybridization assays utilizean oligodeoxynucleotide probe that has been immobilized on a solidsupport; as for example in the reverse dot blot procedure (Saiki, R. K.,Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989) Proc. Natl.Acad. Sci. USA 86, 6230). More recently, arrays of immobilized DNAprobes attached to a solid surface have been developed for sequencing byhybridization (SBH) (Drmanac, R., Labat, I., Brukner, I., andCrkvenjakov, R. (1989) Genomics, 4, 114-128), (Strezoska, Z., Pauneska,T., Radosavljevic, D., Labat, I., Drmanac, R., and Crkvenjakov, R.(1991) Proc. Natl. Acad. Sci. USA, 88, 10089-10093). SBH uses an orderedarray of immobilized oligodeoxynucleotides on a solid support. A sampleof unknown DNA is applied to the array, and the hybridization pattern isobserved and analyzed to produce many short bits of sequence informationsimultaneously. An enhanced version of SBH, termed positional SBH(PSBH), has been developed which uses duplex probes containingsingle-stranded 3′-overhangs. (Broude, N. E., Sano, T., Smith, C. L.,and Cantor, C. R. (1994) Proc. Natl. Acad. Sci. USA, 91, 3072-3076). Itis now possible to combine a PSBH capture approach with conventionalSanger sequencing to produce sequencing ladders detectable, for exampleby gel electrophoresis (Fu, D., Broude, N. E., Köster, H., Smith, C. L.and Cantor, C. R. (1995) Proc. Natl. Acad. Sci. USA 92, 10162-10166).

For the arrays utilized in these schemes, there are a number of criteriawhich must be met for successful performance. For example, theimmobilized DNA must be stable and not desorb during hybridization,washing or analysis. The density of the immobilized oligodeoxynucleotidemust be sufficient for the ensuing analyses. There must be minimalnon-specific binding of the DNA to the surface. In addition, theimmobilization process should not interfere with the ability of theimmobilized probes to hybridize and to be substrates for enzymatic solidphase synthesis. For the majority of applications, it is best for onlyone point of the DNA to be immobilized, ideally a terminus.

In recent years, a number of methods for the covalent immobilization ofDNA to solid supports have been developed which attempt to meet all thecriteria listed above. For example, appropriately modified DNA has beencovalently attached to flat surfaces functionalized with amino acids(Running, J. A., and Urdea, M. S. (1990) Biotechniques, 8, 276-277),(Newton, C. R., et al., (1993) Nucl. Acids. Res., 21, 1155-1162.),(Nikiforov, T. T., and Rogers, Y. H. (1995) Anal. Biochem., 227,201-209), carboxyl groups, (Zhang, Y., et al., (1991) Nucl. Acids. Res.,19 3929-3933), epoxy groups (Lamture, J. B. et al., (1994) Nucl. Acids.Res., 22, 2121-2125), (Eggers, M. D., et al., (1994) BioTechniques, 17,516-524) or amino groups (Rasmussen, S. R., et al., (1991) Anal.Biochem., 198, 138-142). Although many of these methods were quitesuccessful for their respective applications, the density ofoligonucleotide bound (maximum of approximately 20 fmol of DNA persquare millimeter of surface) (Lamture, J. B., et al., (1994) Nucl.Acids. Res. 22, 2121-2125), (Eggers, M. D., et al., (1994)BioTechniques, 17, 516-524), was far less than the theoretical packinglimit of DNA.

Therefore, a method for achieving higher densities of immobilizednucleic acids on a surface is needed. In particular, a method forachieving higher densities of surface immobilized nucleic acids whichpermits use, manipulation and further reaction of the immobilizednucleic acids, as well as analysis of the reactions, is needed.

In connection with the need for improved nucleic acid immobilizationmethods for use, for example, in analytical and diagnostic systems, isthe need to develop sophisticated laboratory tools that will automateand expedite the testing and analysis of biological samples. At theforefront of recent efforts to develop better analytical tools is thegoal of expediting the analysis of complex biochemical structures. Thisis particularly true for human genomic DNA, which is comprised of atleast about one hundred thousand genes located on twenty fourchromosomes. Each gene codes for a specific protein, which fulfills aspecific biochemical function within a living cell. Changes in a DNAsequence are known as mutations and can result in proteins with alteredor in some cases even lost biochemical activities; this in turn cancause a genetic disease. More than 3,000 genetic diseases are currentlyknown. In addition, growing evidence indicates that certain DNAsequences may predispose an individual to any of a number of geneticdiseases, such as diabetes, arteriosclerosis, obesity, certainautoimmune diseases and cancer. Accordingly, the analysis of DNA is adifficult but worthy pursuit that promises to yield informationfundamental to the treatment of many life threatening diseases.

Unfortunately, the analysis of DNA is made particularly cumbersome dueto size and the fact that genomic DNA includes both coding andnon-coding sequences (e.g., exons and introns). As such, traditionaltechniques for analyzing chemical structures, such as the manualpipeting of source material to create samples for analysis, are ofminimal value. To address the scale of the necessary analysis,scientists have developed parallel processing protocols for DNAdiagnostics.

For example, scientists have developed robotic devices that eliminatethe need for manual pipeting and spotting by providing a robotic armthat carries at its proximal end a pin tool device that consists of amatrix of pin elements. The individual pins of the matrix are spacedapart from each other to allow each pin to be dipped within a well of amicrotiter plate. The robotic arm dips the pins into the wells of themicrotiter plate thereby wetting each of the pin elements with samplematerial. The robotic arm then moves the pin tool device to a positionabove a target surface and lowers the pin tool to the surface contactingthe pins against the target to form a matrix of spots thereon.Accordingly, the pin tool expedites the production of samples bydispensing sample material in parallel.

Although this pin tool technique works well to expedite the productionof sample arrays, it suffers from several drawbacks. First during thespotting operation, the pin tool actually contacts the surface of thesubstrate. Given that each pin tool requires a fine point in order thata small spot size is printed onto the target, the continuous contact ofthe pin tool against the target surface will wear and deform the fineand delicate points of the pin tool. This leads to errors which reduceaccuracy and productivity.

An alternative technique developed by scientists employs chemicalattachment of sample material to the substrate surface. In oneparticular process, DNA is synthesized in situ on a substrate surface toproduce a set of spatially distinct and diverse chemical products. Suchtechniques are essentially photolithographic in that they combine solidphase chemistry, photolabile protecting groups and photo activatedlithography. Although these systems work well to generate arrays ofsample material, they are chemically intensive, time consuming, andexpensive.

It is further troubling that neither of the above techniques providesufficient control over the volume of sample material that is dispensedonto the surface of the substrate. Consequently, error can arise fromthe failure of these techniques to provide sample arrays with wellcontrolled and accurately reproduced sample volumes. In an attempt tocircumvent this problem, the preparation process will often dispensegenerous amounts of reagent materials. Although this can ensuresufficient sample volumes, it is wasteful of sample materials, which areoften expensive and of limited availability.

Even after the samples are prepared, scientists still must confront theneed for. sophisticated diagnostic methods to analyze the preparedsamples. To this end, scientists employ several techniques foridentifying materials such as DNA. For example, nucleic acid sequencescan be identified by hybridization with a probe which is complementaryto the sequence to be identified. Typically, the nucleic acid fragmentis labeled with a sensitive reporter function that can be radioactive,fluorescent, or chemiluminescent. Although these techniques can workwell, they do suffer from certain drawbacks. Radioactive labels can behazardous and the signals they produce decay over time. Nonisotopic(e.g. fluorescent) labels suffer from a lack of sensitivity and fadingof the signal when high intensity lasers are employed during theidentification process. In addition, labeling is a laborious and timeconsuming error prone procedure. Consequently, the process of preparingand analyzing arrays of a biochemical sample material is complex anderror prone.

Therefore, it is an object herein to provide improved systems andmethods for preparing arrays of sample material. It is a further objectto provide systems that allow for the rapid production of sample arrays.It is a further object herein to provide supports to which highdensities of nucleic acids molecules are linked.

SUMMARY OF THE INVENTION

Processes for immobilizing a high density of nucleic acids on a surface,which are based on rapidly reacting a free thiol group of a modifiedsurface or modified nucleic acid, under appropriate conditions, with athiol-reactive functionality of the other component (surface or nucleicacid) are provided. This reaction may be direct or through abifunctional cross-linking reagent. In a preferred embodiment, themodified nucleic acid includes a thiol group and the cross-linkingreagent contains an iodoacetyl group.

Solid supports to which are linked “beads” which are linked to nucleicacid molecules are also provided. The beads are not necessarilyspherical, but refer to particles that are conjugated to the solidsupport to thereby increase the surface area of the solid support and/orto provide an alternative surface for conjugation of nucleic acids orother molecules. The beads are preferably of a size of about 1 μm to 100μm. Compositions containing at least one bead conjugated to a solidsupport and further conjugated to at least one molecule, particularly anucleic acid are provided. The bead is formed from any suitable matrixmaterial known to those of skill in the art, including those that areswellable and nonswellable. The solid support is any support known tothose of skill in the art for use as a support matrix in chemicalsyntheses and analyses. In such instances, the nucleic acid is linked tothe “bead” via a sulfur atom as described herein. In certainembodiments, the beads may be conjugated on the solid support in wellsor pits on the surface, or the beads may be arranged in the form of anarray on the support.

Preferably the bead is made of a material selected from materials thatserve as solid supports for synthesis and for assays including but notlimited to: silica gel, glass, magnet, polystyrene/1% divinylbenzeneresins, such as Wang resins, which are Fmoc-aminoacid-4-(hydroxy-methyl)phenoxymethylcopoly(styrene-1% divinylbenzene(DVD)) resin, chlorotrityl (2-chlorotritylchloride copolystyrene-DVBresin) resin, Merrifield (chloromethylated copolystyrene-DVB) resinmetal, plastic, cellulose, cross-linked dextrans, such as those soldunder the tradename Sephadex (Pharmacia) and agarose gel, such as gelssold under the tradename Sepharose (Pharmacia), which is a hydrogenbonded polysaccharide-type agarose gel, and other such resins and solidphase supports known to those of skill in the art. In a preferredembodiment, the bead is of a size in the range of about 0.1 to 500 μm,more preferably about 1 to 100 μm, in diameter.

The solid support is in any desired form, including, but not limited to:a bead, capillary, plate, membrane, wafer, comb, pin, a wafer with pits,an array of pits or nanoliter wells and other geometries and forms knownto those of skill in the art.

In another aspect, kits for immobilized nucleic acids on an insolublesupport are provided. In one embodiment, the kit can comprise anappropriate amount of: i) a thiol-reactive cross-linking reagent; andii) a surface-modifying reagent for modifying a surface withfunctionality which can react with the thiol-reactive cross-linkingreagent. The kit can optionally include an insoluble support, e.g., asolid surface, magnetic microbeads or silicon wafers, for use inimmobilizing nucleic acids. The kit can also optionally includeappropriate buffers as well as instructions for use.

Use of these processes for immobilizing nucleic acid molecules onto asolid support results in at least 12.5-fold higher immobilization thanpreviously reported techniques. The processes are therefore particularlyuseful for forming nucleic acid launching pads for mass spectrometry.

The nucleic acids immobilized on a surface using the methods providedherein can be used in a variety of solid phase nucleic acid chemistryapplications, including but not limited to nucleic acid synthesis(chemical and enzymatic), hybridization and/or extension, and indiagnostic methods based in nucleic acid detection and polymorphismanalyses (see, e.g., U.S. Pat. No. 5,605,798). Accordingly, furtherprovided herein are methods of reacting nucleic acid molecules in whichthe nucleic acid molecules are immobilized on a surface either byreacting a thiol-containing derivative of the nucleic acid molecule withan insoluble support containing a thiol-reactive group or by reacting athiol-containing insoluble support with a thiol-reactivegroup-containing derivative of the nucleic acid molecule and thereafterfurther reacting the immobilized nucleic acid molecules.

In a particular embodiment of the methods of reacting immobilizednucleic acids, the immobilized nucleic acid is further reacted byhybridizingxwith a nucleic acid that is complementary to the immobilizednucleic acid or a portion thereof. Such hybridization reactions can beused to detect the presence of a specific nucleic acid in a sample. Thisis of particular use in the detection of pathogens in a sample, such asa biological sample, that may be employed in the diagnosis of diseases.

Therefore, also provided herein are methods of detecting a targetnucleic acid in a sample wherein a thiol-containing nucleic acidcomplementary to the target nucleic acid is immobilized to a surfaceusing the processes described herein and the sample is contacted withthe surface under conditions whereby target nucleic acid in the samplehybridizes to the immobilized nucleic acid. The hybridized targetnucleic acid may be detected using a variety of methods, the preferredmethod being mass spectrometry. Further provided herein are methods ofdetecting alterations (e.g., deletions, insertions and conversions) inthe nucleotide sequence of the target nucleic acid. In these methods,the molecular weight of the hybridized target nucleic acid, asdetermined by mass spectrometry, is compared to the molecular weightexpected for the target nucleic acid sequence. Deviations of themeasured molecular weight from the expected molecular weight areindicative of an alteration in the nucleotide sequence of the targetnucleic acid.

In other methods of detecting a target nucleic acid in a sample asprovided herein, the target nucleic acid is immobilized to a surfacecontaining thiol-reactive groups. In these methods, prior toimmobilization, the target nucleic acid is amplified in a reaction inwhich an oligonucleotide primer contains a 3′- or 5′-disulfide linkageand the resulting product is reduced to generate a thiol-containingnucleic acid. The thiol-containing nucleic acid is immobilized to asurface containing thiol-reactive groups and is contacted with asingle-stranded nucleic acid that is complementary to the immobilizednucleic acid or a portion thereof. Hybridization of the single-strandednucleic acid may be detected by a variety of methods. For example, thesingle-stranded nucleic acid may be labeled with a readily detectablemoiety, e.g., radioactive or chemiluminescent labels. In a preferredembodiment, the single-stranded nucleic acid is detected by massspectrometry.

In another embodiment of the methods of reacting immobilized nucleicacids, the immobilized nucleic acid is further reacted by extension of anucleic acid that is hybridized to the immobilized nucleic acid or aportion thereof. Extension reactions such as these can be used, forexample, in methods of sequencing DNA molecules that are immobilized toan insoluble support using the processes described herein. Thus, alsoprovided herein are methods of determining the sequence of a DNAmolecule on a substrate in which a thiol-containing derivative of theDNA molecule is immobilized on the surface of an insoluble supportcontaining thiol-reactive groups and hybridized with a single-strandednucleic acid complementary to a portion of the immobilized DNA moleculeprior to carrying out DNA synthesis in the presence of one or moredideoxynucleotides.

Extension of a nucleic acid primer that is hybridized to a nucleic acidimmobilized to a surface as provided herein also can be used in thedetection of nucleotide sequence alterations (e.g., deletions,insertions, conversions) of a target nucleic acid. Accordingly, providedherein are methods of detecting alterations in a target nucleic acidsequence in which a single-stranded nucleic acid is hybridized to athiol-containing target nucleic acid immobilized to a solid supportaccording to the processes provided herein and the hybridizedsingle-stranded nucleic acid is extended by addition of nucleotides tothe 3′ end of the molecule. The extension product is characterized by,for example, mass spectrometry to determine whether its characteristicsdiffer from those expected of a sequence complementary to theimmobilized target nucleic acid. Thus, for example, the molecular weightof the extension product determined by mass spectrometry is compared tothe expected molecular weight of a nucleic acid complementary to thetarget nucleic acid. Deviations from the expected molecular weight areindicative of an alteration in the sequence of the target nucleic acid.

In particular embodiments of the methods of detecting alterations in atarget nucleic acid sequence provided herein, the target nucleic acidmay be amplified prior to immobilization to a thiol-reactive surface ina reaction in which an oligonucleotide primer contains a 3′- or5′-disulfide linkage. The resulting product is reduced to generate athiol-containing target nucleic acid. The thiol-containing targetnucleic acid is then immobilized to a surface containing thiol-reactivegroups and the single-stranded complementary nucleic acid is hybridizedthereto and extended.

In a further embodiment of the methods of detecting alterations in atarget nucleic acid sequence provided herein, a single-stranded nucleicacid complementary to the target nucleic acid is immobilized to asurface through a linkage that includes a thiol group-thiol reactivefunctionality bond and a cleavable linker moiety. The sample containingtarget nucleic acid is contacted with the surface under conditionswhereby the target hybridizes with the immobilized single-strandednucleic acid. The immobilized single-stranded nucleic acid is extendedby addition of nucleotides to the 3′ end of the molecule. Followingextension, the double-stranded molecule is denatured and thesingle-stranded immobilized extension product is cleaved from thesurface at the position of the linker. The extension product ischaracterized by, for example, mass spectrometry to determine whetherits characteristics differ from those expected of a sequencecomplementary to the immobilized target nucleic acid.

It is understood that all applications of the solid phase nucleic acidchemistry based on nucleic acids immobilized to a solid substrateaccording to the processes provided herein can be conducted withthiol-containing nucleic acids and a thiol-reactive surface as well aswith thiol-reactive nucleic acids and a thiol-containing support.

Methods of forming an array of nucleic acids on a surface of a substrateby contacting thiol-containing nucleic acids with an insoluble supportcontaining thiol-reactive groups positioned in an ordered arrangement onthe surface of the support are also provided herein. In an alternativemethod of forming an array of nucleic acids on a surface of a substrateas provided herein, an insoluble support containing thiolfunctionalities positioned in an ordered arrangement on the surface ofthe support is contacted with nucleic acids containing a thiol-reactivegroup.

Further provided herein are systems and methods for preparing a samplefor analysis, and more specifically to systems and methods fordispensing low volumes of fluid material onto a substrate surface forgenerating an array of samples for diagnostic analysis. Systems andmethods provided herein for preparing arrays of sample material aregenerally less expensive to employ and conserve reagent materials whileallowing for the rapid production of highly reproducible sample arrays.

Provided herein with respect to systems and methods for dispensing lowvolumes of fluid material onto a substrate surface are serial andparallel dispensing tools that can be employed to generate multi-elementarrays of sample material on a substrate surface. The substrate surfacescan be flat or geometrically altered to include wells of receivingmaterial.

In one embodiment, the tool is one that allows the parallel developmentof a sample array. To this end, the tool can be understood as anassembly of vesicle elements, or pins, wherein each of the pins caninclude a narrow interior chamber suitable for holding nanoliter volumesof fluid. Each of the pins can fit inside a housing that itself has aninterior chamber. The interior housing can be connected to a pressuresource that will control the pressure within the interior housingchamber to regulate the flow of fluid through the interior chamber ofthe pins. This allows for the controlled dispensing of defined volumesof fluid from the vesicles.

In an alternative embodiment, the tool includes a jet assembly that caninclude a capillary pin having an interior chamber, and a transducerelement mounted to the pin and capable of driving fluid through theinterior chamber of the pin to eject fluid from the pin. In this way,the tool can dispense a spot of fluid to a substrate surface by sprayingthe fluid from the pin. Alternatively, the transducer can cause a dropof fluid to extend from the capillary so that fluid can be passed to thesubstrate by contacting the drop to the surface of the substrate.

Further, the tool can form an array of sample material by dispensingsample material in a series of steps, while moving the pin to differentlocations above the substrate surface to form the sample array. In afurther embodiment, the prepared sample arrays are passed to a plateassembly that disposes the sample arrays for analysis by massspectrometry. To this end, a mass spectrometer is provided thatgenerates a set of spectra signal which can be understood as indicativeof the composition of the sample material under analysis.

In one aspect, the dispensing apparatus provided herein for dispensingdefined volumes of fluid, including nanovolumes and sub-nanovolumes offluid, in chemical or biological procedures onto the surface of asubstrate can include a housing having a plurality of sides and a bottomportion having formed therein a plurality of apertures, the walls andbottom portion of the housing defining an interior volume; one or morefluid transmitting vesicles, or pins, mounted within the apertures,having a nanovolume sized fluid holding chamber for holding nanovolumesof fluid, the fluid holding chamber being disposed in fluidcommunication with the interior volume of the housing, and a dispensingelement that is in communication with the interior volume of the housingfor selectively dispensing nanovolumes of fluid from the nanovolumesized fluid transmitting vesicles when the fluid is loaded into thefluid holding chambers of the vesicles. As described herein, this allowsthe dispensing element to dispense nanovolumes of the fluid onto thesurface of the substrate when the apparatus is disposed over and inregistration with the substrate.

In one embodiment the fluid transmitting vesicle has an open proximalend and a distal tip portion that extends beyond the housing bottomportion when mounted within the apertures. In this way the open proximalend can dispose the fluid holding chamber in fluid communication withthe interior volume when mounted with the apertures. Optionally, theplurality of fluid transmitting vesicles are removably and replaceablymounted within the apertures of the housing, or alternatively caninclude a glue seal for fixedly mounting the vesicles within thehousing.

In one embodiment the fluid holding chamber includes a narrow boredimensionally adapted for being filled with the fluid through capillaryaction, and can be sized to fill substantially completely with the fluidthrough capillary action.

In one embodiment, the plurality of fluid transmitting vesicles comprisean array of fluid delivering needles, which can be formed of metal,glass, silica, polymeric material, or any other suitable material.

In one embodiment the housing can include a top portion, and mechanicalbiasing elements for mechanically biasing the plurality of fluidtransmitting vesicles into sealing contact with the housing bottomportion. In one particular embodiment, each fluid transmitting vesiclehas a proximal end portion that includes a flange, and further includesa seal element disposed between the flange and an inner surface of thehousing bottom portion for forming a seal between the interior volumeand an external environment. The biasing elements can be mechanical andcan include a plurality of spring elements each of which is coupled atone end to the proximal end of each of the plurality of fluidtransmitting vesicles, and at another end to an inner surface of thehousing top portion. The springs can apply a mechanical biasing force tothe vesicle proximal end to form the seal.

In a further embodiment, the housing further includes a top portion, andsecuring element for securing the housing top portion to the housingbottom portion. The securing element can comprise a plurality offastener-receiving apertures formed within one of the top and bottomportions of the housing, and a plurality of fasteners for mountingwithin the apertures for securing together the housing top and bottomportions.

In one embodiment the dispensing element can comprise a pressure sourcefluidly coupled to the interior volume of the housing for disposing theinterior volume at a selected pressure condition. Moreover, in anembodiment wherein the fluid transmitting vesicles are filled throughcapillary action, the dispensing element can include a pressurecontroller that can vary the pressure source to dispose the interiorvolume of the housing at varying pressure conditions. This allows thecontroller varying element to dispose the interior volume at a selectedpressure condition sufficient to offset the capillary action to fill thefluid holding chamber of each vesicle to a predetermined heightcorresponding to a predetermined fluid amount. Additionally, thecontroller can further include a fluid selection element for selectivelydischarging a selected nanovolume fluid amount from the chamber of eachvesicle. In one particular embodiment, a pressure controller is includedthat operates under the controller of a computer program operating on adata processing system to provide variable control over the pressureapplied to the interior chamber of the housing.

In one embodiment the fluid transmitting vesicle can have a proximal endthat opens onto the interior volume of the housing, and the fluidholding chamber of the vesicles are sized to substantially completelyfill with the fluid through capillary action without forming a meniscusat the proximal open end. Optionally, the apparatus can have pluralvesicles, wherein a first portion of the plural vesicles include fluidholding chambers of a first size and a second portion including fluidholding chambers of a second size, whereby plural fluid volumes can bedispensed.

In another embodiment, the dispensing apparatus can include a fluidselection element that has a pressure source coupled to the housing andin communication with the interior volume for disposing the interiorvolume at a selected pressure condition, and an adjustment element thatcouples to the pressure source for varying the pressure within theinterior volume of the housing to apply a positive pressure in the fluidchamber of each of the fluid transmitting vesicles to vary the amount offluid dispensed therefrom. The selection element and adjustment elementcan be computer programs operating on a data processing system thatdirects the operation of a pressure controller connected to the interiorchamber.

In a further alternative embodiment, the apparatus provided herein isfor dispensing a fluid in chemical or biological procedures into one ormore wells of a multi-well substrate. The apparatus can include ahousing having a plurality of sides and a bottom portion having formedtherein a plurality of apertures, the walls and bottom portion definingan interior volume, a plurality of fluid transmitting vesicles, mountedwithin the apertures, having a fluid holding chamber disposed incommunication with the interior volume of the housing, and a fluidselection and dispensing means in communication with the interior volumeof the housing for variably selecting am amount of the fluid loadedwithin the fluid holding chambers of the vesicles to be dispensed from asingle set of the plurality of fluid transmitting vesicles. Accordingly,the dispensing means dispenses a selected amount of the fluid into thewells of the multi-well substrate when the apparatus is disposed overand in registration with the substrate.

In yet another embodiment, provided herein is a fluid dispensingapparatus for dispensing fluid in chemical or biological procedures intoone or more wells of a multi-well substrate, that comprises a housinghaving a plurality of sides and top and bottom portions, the bottomportion having formed therein a plurality of apertures, the walls andtop and bottom portions of the housing defining an interior volume, aplurality of fluid transmitting vesicles, mounted within the apertures,having a fluid holding chamber sized to hold nanovolumes of the fluid,the fluid holding chamber being disposed in fluid communication with thevolume of the housing, and mechanical biasing element for mechanicallybiasing the plurality of fluid transmitting vesicles into sealingcontact with the housing bottom portion.

General methods for preparing an array of sample material on a surfaceof a substrate as described herein include the steps of providing avesicle having an interior chamber containing a fluid, disposing thevesicle adjacent a first location on the surface of the substrate,controlling the vessel for delivering a nanoliter volume of a fluid atthe first location of the surface of the substrate, and moving thevesicle to a set of positions adjacent to the surface substrate wherebyfluid is dispensed at each location of the set of positions for formingan array of sample material;

Substrates employed during the general processes of preparing an arrayof sample material described herein can include flat surfaces forreceiving the sample material as well as having the surfaces thatinclude wells formed on the surface for defining locations for receivingthe fluid that can be ejected from the chambers of the vesicles. Suchsubstrates can be silicon, metal, plastic, a membrane, polymericmaterial, a metal-grafted polymer, as well as a substrate that isfunctionalized chemically, functionalized with beads, functionalizedwith dendrite trees of captured material, or any combinations of theabove or any similar suitable material for receiving the dispensedfluid.

It is understood that in the general methods for preparing an array ofsample material on a substrate surface described herein the apparatuscan dispense both an analyte material as well as a support material,such as a matrix material, that aids in the analysis of the analyte. Tothis end the methods provided herein can include the steps of depositinga matrix material onto the substance of the substrate. Further themethods can also include a step of waiting a predetermined period oftime to allow a solvent of the matrix material to evaporate. Once thesolvent of the matrix material has evaporated, the methods herein caninclude a step of ejecting a volume of analyte fluid into the evaporatedmatrix material to dissolve with the matrix material and to form acrystalline structure on the substrate surface. It is understood thatthis step of redissolving the matrix material with the analyte materialaids in the analysis of the composition of the material during certainanalytical processes, such as mass spectrometry.

In an alternative practice, the methods herein can include a step ofdispensing a mixture that consists of the analyte material and thematrix material, as well as other material compositions. In this way thematrix and the analyte are delivered to the surface of the substrate asone volume of material. In a further step, the prepared arrays of samplematerial can be provided to a diagnostic tool for determininginformation that is representative of the composition of the samplematerial.

Once such diagnostic tool can include a mass spectrometer. The massspectrometers can be time of flight mass spectrometers, Fouriertransform mass spectrometers or any other suitable type of massspectrometer that allows the analysis of composition of the samplearray.

In one practice of the methods, the step of providing a vesicle havingan interior chamber includes the step of providing a vesicle having apiezo electric element for causing fluid to move through the chamber.This method can also include the step of moving the vesicle byrasterizing the vesicle across the surface of the substrate, to form thearray of sample material.

In an alternative practice of the methods, parallel processing protocolscan be employed wherein the vesicle that is employed during theprocessing includes a vesicle assembly that has a plurality of vesiclesarranged into a matrix for dispensing fluid to a first plurality oflocations on the substrate surface. In this way in a single operation,the method provides for forming a matrix of a sample material on thesubstrate surface. Offset printing can also be employed to form a largematrix of sample material by employing multiple printing steps with thevesicle matrix. Other printing techniques can be employed by the presentinvention without departing from the scope thereof.

In another embodiment, fluid can be dispensed to the surface of thesubstrate by contacting the vesicle against the surface of the substrateto spot the surface of the substrate with sample material.Alternatively, the methods provide for another non-contact printingapproach wherein the processes of the invention cause a drop of fluid tobe formed on at the distal tip of the vesicle. It is the drop of fluidthat is contacted against the surface of the substrate for deliveringsampling material thereto. This provides for the controlled delivery forthe known volume of fluid without resulting in the contacting of thevesicle against the surface of the substrate.

In further embodiments, vesicles are provided having an interior chamberthat is dimensionally adapted to allow filling of the chamber bycapillary action.

In another aspect, methods are provided for analyzing a material, thatcomprise the steps of providing a vesicle suitable for carrying a fluidhaving the material therein, disposing the vesicle adjacent a firstlocation of the surface of the substrate, controlling the vesicle todeliver a nanoliter volume of the fluid to provide a defined andcontrolled volume of fluid at the first location of the surface of thesubstrate, moving the vesicle to a second position adjacent a secondlocation on the surface on the substrate to dispense a defined andcontrolled volume of the material along an array of locations along thesubstrate surface, and performing mass spectrometry analysis of thematerial at each location of the array. These methods can include thestep of mixing a matrix material and an analyte material to form thefluid being delivered to the substrate surface. Alternatively, thisembodiment can include the steps of filling a chamber contained withinthe vesicle with a matrix material and dispensing the matrix material tothe array of locations. Subsequently, analyte can be dispensed. The stepof performing mass spectrometry can include the step of performing amatrix assisted laser desorption ionization mass spectrometry, as wellas time of flight mass spectrometry, or a Fourier transformspectrometry.

In another aspect, apparatus for forming an array of a sample materialon a surface of a substrate are provided. Such apparatus will compromisea vesicle having a distal end suitable for carrying a fluid thereon, amovable arm having a distal portion mounted to the vesicle, a controllerfor moving the arm to dispose the vesicle adjacent a first location onthe surface on the substrate and for controlling the vesicle to providea nanoliter volume of the fluid at the first location of the surface ofthe substrate, and a diagnostic tool for analyzing the material togenerate a composition signal that is representative of the chemicalcomposition of the material. In this apparatus the vesicle cancompromise a solid shaft of material as well as a vesicle having aninterior chamber suitable for carrying fluid as well as a chamber forcarrying a fluid in a transducer element for ejecting fluid from thatchamber.

Further provided herein are substrates having a surface for carrying anarray of a matrix material and formed according to a process comprisingthe steps of a providing a vesicle suitable for transferring a fluidcontaining a matrix material, disposing the vesicle adjacent a firstlocation on the surface on the substrate, controlling the vesicle todeliver the fluid to the first location of the surface of the substrate,and moving a vesicle to a set of positions adjacent the surface of thesubstrate and delivering fluid at each of these locations to form anarray of matrix material. This substrate itself can be a flat siliconchip as well as a any other suitable material, and can be pitted,include wells, and have wells that have rough interior surfaces.

In particular embodiments, the methods of forming an array of nucleicacids on a surface of a substrate as provided herein include contactingpredetermined positions of the surface of an insoluble support withthiol-containing nucleic acid solutions dispensed to the positions witha vesicle having an interior chamber containing the respective solutionswhereby the predetermined positions incorporate thiol-reactive groups.Alternatively, the entire surface of the substrate is derivatized withthe thiol-reactive groups and thiol-containing nucleic acid is dispensedto predetermined positions on the surface in an array-forming manner.Also provided herein are substrates having a surface carrying an arrayof nucleic acids formed by the methods described herein.

The above and further features and advantages of the instant inventionwill become clearer from the following Figures, Detailed Description andClaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a system for preparing arrays of a sample materialfor analysis.

FIG. 2 illustrates a pin assembly suitable for use with the systemdepicted in FIG. 1 for implementing a parallel process of dispensingmaterial to a surface of a substrate.

FIG. 3 depicts a bottom portion of the assembly shown in FIG. 2.

FIG. 4 depicts an alternative view of the bottom portion of the pinassembly depicted in FIG. 2.

FIGS. 5A-5D depict a method for preparing an array of sample material.

FIGS. 6A-6B depict an alternative assembly for dispensing material tothe surface of a substrate.

FIG. 7 is a schematic showing covalent attachment ofoligodeoxynucleotides to a silicon dioxide surface as described in themethods herein. In particular, silicon dioxide was reacted with3-aminopropyltriethoxysilane to produce a uniform layer of primary aminogroups on the surface. A heterobifunctional crosslinking agent was thenreacted with the primary amine to incorporate an iodoacetamide-group. Anoligodeoxynucleotide containing a 3′- or 5′-disulfide (shown as the 5′)was treated with tris-(2-carboxyethyl) phosphine (TCEP) to reduce thedisulfide to a free thiol, which was then coupled to theiodoacetamido-surface.

FIG. 8 is a graph which plots conjugation of oligodeoxynucleotide probesto a silicon surface as a function of TCEP concentration used in thedisulfide reduction.

FIG. 9 is a matrix assisted laser desorbtionlionization-time-of-flight(MALDI-TOF) mass spectrum of a silicon wafer with theoligodeoxynucleotide sequence denoted “TCUC”(5′-GAATTCGAGCTCGGTACCCGG-3′; SEQ ID NO 1) covalently bound essentiallyas described in FIG. 7 and the oligodeoxynucleotide sequence denoted“MJM6” (5′-CCGGGTACCGAGCTCGAATTC-3′; SEQ ID NO 2) hybridized thereto.

FIG. 10 is a schematic of the immobilization of specificthiol-containing DNA targets generated by polymerase chain reaction(PCR) to the surface of a silicon wafer. An oligonucleotide [SEQ ID NO:7] complementary to a portion of the DNA target sequence was hybridizedto the immobilized DNA target and MALDI-TOF MS analysis was performedrevealing a predominant signal at an observed mass-to-charge ratio of3618.33 corresponding to the hybridized oligonucleotide, which has a thetheoretical mass-to-charge ratio of 3622.4.

FIG. 11 depicts one embodiment of a substrate having wells etchedtherein that are suitable for receiving material for analysis.

FIG. 12 depicts one example of spectra obtained from a linear time offlight mass spectrometer instrument and representative of the materialcomposition of the sample material on the surface of the substratedepicted in FIG. 11.

FIG. 13 depicts molecular weights determined for the sample materialhaving spectra identified in FIG. 12.

FIG. 14 is a schematic of a 4×4 (16-location) DNA array on the surfaceof a silicon wafer with the thiol-containing oligonucleotide moleculesdenoted “Oligomer 1”, [5′-CTGGATGCGTCGGATCATCTTTTTT-(S)-3′; SEQ ID NO:8], Oligomer 2 [5′-(S)-CCTCTTGGGAACTGTGTAGTATT-3′; SEQ ID NO: 3]and“Oligomer 3” (SEQ ID NO: 1; a free thiol derivative “TCUC”oligonucleotide of EXAMPLE 1) covalently bound to 16 locations on thesurface of the silicon wafer essentially as described in EXAMPLE 2.

FIG. 15 is a schematic of the hybridization of specific oligonucleotidesto each of the 16 locations of the DNA hybridization array of FIG. 14with the Oligomer 1 complementary oligonucleotide(5′-GATGATCCGACGCATCAGAATGT-3′; SEQ ID NO: 9) bound to Oligomer 1, theOligomer 2 complementary oligonucleotide (5′-AATACTACACAG-3′; SEQ ID NO:7) bound to Oligomer 2 and the Oligomer 3 complementary oligonucleotide(5′-CCGGGTACCGAGCTCGAATTC-3′; SEQ ID NO: 2) bound to Oligomer 3.

FIG. 16 is a representative MALDI-TOF mass spectrum of a 4×4(16-location) DNA array on a silicon wafer shown schematically in FIG.15. The spectrum reveals a single, predominant signal of an experimentalmass-to-charge ratio in each location corresponding to the specifichybridized oligonucleotides. The 2+ indicates the position of a doublycharged molecule used as a reference standard during MALDI-TOF MSanalysis. The * denotes residual amounts of contaminatingoligonucleotide that remain on the surface of the chip following washingprocedures. The relative position of the * signal reveals theapproximate size of the contaminating oligonucleotide.

FIG. 17 is a representative MALDI-TOF mass spectrum of an 8×8(64-location) DNA array. The spectrum reveals a single, predominantsignal of an experimental mass-to-charge ratio corresponding to thepredicted specific hybridized oligonucleotides. The * denotes residualamounts of contaminating oligonucleotide that remain on the surface ofthe wafer following washing procedures. The relative position of the *signal reveals the approximate size of the contaminatingoligonucleotide.

FIG. 18 is an illustration of nucleotide extension of a DNA primerannealed to a thiol-containing DNA template immobilized to the surfaceof a SIAB-derivatized silicon wafer. A complementary 12-meroligonucleotide primer [SEQ ID NO: 12] was hybridized to a 27-merthiol-containing oligonucleotide [SEQ ID NO: 11] immobilized to asilicon support through the SIAB crosslinker. The silicon surfacecontaining the immobilized DNA duplex was incubated with DNA polymerasein the presence of dATP, dCTP, dGTP and ddTTP under extension conditionsand subjected to MALDI-TOF MS analysis. The mass spectrum of the siliconwafer revealed the presence of two predominant signals; one of amass-to-charge ratio equal to the unextended 12-mer oligonucleotide aswell as a signal corresponding to a 15-mer DNA molecule that has beenextended on the wafer by 3 nucleotides to the first position in thesequence in which a ddTTP was incorporated.

FIG. 19 diagrams an experiment designed to test the effect of thedistance between the SIAB-derivatized surface and the DNA duplex formedon primer extension reactions. Two thiol-containing oligonucleotides ofdifferent sequence [SEQ ID NOs: 8 & 11] were immobilized to aSIAB-derivatized silicon surface and incubated with specificoligonucleotides that form a DNA duplex with 0, 3, 6, 9 and 12 basespacers between the SIAB-derivatized surface and the DNA duplex formedby the oligonucleotide hybridized to the immobilized thiol-containingDNA. The free 3′-end of the hybridized oligonucleotide was extendedusing either Sequenase DNA polymerase or ThermoSequenase DNA polymerasein the presence of the three deoxynucleotide triphosphates and thecorresponding diddeoxynucleotide triphosphate under extension conditionsand the resulting reaction products were subjected to MALDI-TOF MSanalysis.

FIG. 20 is a representative MALDI-TOF mass spectrum of the specificextension products of the primer extension experiment illustrated inFIG. 19. The spectra in the left-hand column are those resulting fromMALDI-TOF MS analysis of the extension reactions in which Sequenase wasused. The spectra in the right-hand column are those resulting fromanalysis of the extension reactions in which ThermoSequenase was used.ThermoSequenase DNA polymerase was able to extend the 3′-end of thehybridized DNA primer where the distance between the DNA duplex and thesurface of the derivatized silicon wafer varied between 0 to 12nucleotides. Sequenase DNA polymerase also was able to extend thehybridized DNA where the distance between the DNA duplex and the siliconwafer was between 3 and 9 nucleotides.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference herein.

As used herein, the term, “nucleic acid” refers to oligonucleotides orpolynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) as well as analogs of either RNA or DNA, for example, made fromnucleotide analogs, any of which are in single or double-stranded form.Nucleic acid molecules can be synthetic or can be isolated from aparticular biological sample using any number of procedures which arewell-known in the art, the particular procedure chosen being appropriatefor the particluar biological sample.

As used herein nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides such asphosphorothioate nucleotides and deazapurine nucleotides. A complete setof chain-elongating nucleotides refers to four different nucleotidesthat can hybridize to each of the four different bases comprising theDNA template.

As used herein, nucleic acid synthesis refers to any process by whicholigonucleotides or polynucleotides are generated, including, but notlimited to processes involving chemical or enzymatic reactions.

As used herein, the term “array” refers to an ordered arrangement ofmembers or positions. The array may contain any number of members orpositions and can be in any variety of shapes. In preferred embodiments,the array is two-dimensional and contains n×m members, wherein m and nare integers that can be the same or different. In particularlypreferred embodiments, n and m are each 4 or a multiple thereof.

The term “cross-linking agent” is art-recognized, and, as used herein,refers to reagents which can immobilize a nucleic acid to aninsoluble-support, preferably through covalent bonds. Thus, appropriate“cross-linking agents” for use herein includes a variety of agents thatare capable of reacting with a functional group present on a surface ofthe insoluble support and with a functional group present in the nucleicacid molecule. Reagents capable of such reactivity include homo- andhetero-bifunctional reagents, many of which are known in the art.

Heterobifunctional Reagents are Preferred.

As used herein, the term “thio-reactive functionality,” refers to afunctionality which is capable of rapid reaction with a nucleophilicthiol moiety to produce a covalent bond (e.g., a disulfide or thioetherbond). In general, thiol groups are good nucleophiles, and preferredthiol-reactive functionalities are reactive electrophiles. A variety ofthiol-reactive functionalities are known in the art, and include, forexample, haloacetyls (preferably iodoacetyl), diazoketones, epoxyketones, α, β-unsaturated carbonyls (e.g., α, β-enones) and otherreactive Michael acceptors (including maleimide), acid halides, benzylhalides, and the like. In certain embodiments, a free thiol group of adisulfide can react with a free thiol group (i.e., by disulfide bondformation, including by disulfide exchange). A “thiol-reactive”cross-linking agent, as used herein, refers to a cross-linking reagent(or surface) which includes, or can be modified to include, at least onethiol-reactive functionality. It will be understood that reaction of athiol group can be temporarily prevented by blocking with an appropriateprotecting group, as is conventional in the art (see e.g., T. W. Greeneand P. G. M. Wuts “Protective Groups in Organic Synthesis,” 2nd ed. JohnWiley & Sons, (1991)).

As used herein, a selectively cleavable linker is a linker that iscleaved under selected conditions, such as a photocleavable linker, achemically cleavable linker and an enzymatically cleavable linker (i.e.,a restriction endonuclease site or a ribonucleotide/RNase digestion).The linker is interposed between the support and immobilized DNA.

As used herein, the terms “protein”, “polypeptide” and “peptide” areused interchangeably when referring to a translated nucleic acid (e.g. agene product).

As used herein, “sample” shall refer to a composition containing amaterial to be detected. In a preferred embodiment, the sample is a“biological sample” (i.e., any material obtained from a living source(e.g. human, animal, plant, bacteria, fungi, protist, virus). Thebiological sample can be in any form, including solid materials (e.g.tissue, cell pellets and biopsies) and biological fluids (e.g. urine,blood, saliva, amniotic fluid and mouth wash (containing buccal cells)).Preferably solid materials are mixed with a fluid.

As used herein, “substrate” shall mean an insoluble support onto which asample is deposited according to the materials as described herein.Examples of appropriate substrates include beads. (e.g., silica gel,controlled pore glass, magnetic, Sephadex/Sepharose, cellulose),capillaries, flat supports such as glass fiber filters, glass surfaces,metal surfaces (steel, gold, silver, aluminum, copper and silicon),plastic materials including multiwell plates or membranes (e.g., ofpolyethylene, polypropylene, polyamide, polyvinylidenedifluoride), pins(e.g., arrays of pins suitable for combinatorial synthesis or analysisor beads in pits of flat surfaces such as wafers (e.g., silicon wafers)with or without plates.

In the particular methods of immobilizing nucleic acids to a substrateprovided herein, preferred substrates are those which can supportlinkage of nucleic acids thereto at high densities, preferrably suchthat the covalently bound nucleic acids are present on the substrate ata density of at least about 20 fmol/mm², more preferably at least about75 fmol/mm², still more preferably at least about 85 fmol/mm², yet morepreferably at least about 100 fmol/mm², and most preferably at leastabout 150 fmol/mm². Among the most preferred substrates for use in theparticular methods of immobilizing nucleic acids to substrates providedherein is silicon, whereas less preferred substrates include polymericmaterials such as polyacrylamide. Substrates for use in methods ofproducing arrays provided herein include any of a wide variety ofinsoluble support materials including, but not limited to silica gel,controlled pore glass, cellulose, glass fiber filters, glass surfaces,metal surfaces (steel, gold, silver, aluminum, silicon and copper),plastic materials (e.g., of polyethylene, polypropylene, polyamide,polyvinyidenedifluoride) and silicon.

High Density Immobilization of Nucleic Acids to Solid Supports

The methods described herein provide for high density immobilization ofnucleic acid molecules on a insoluble (e.g., solid) support. In general,nucleic acid molecules are immobilized on the insoluble support eitherdirectly or by means of cross-linking agents.

In embodiments of the methods in which a cross-linking reagent is notemployed, a modified nucleic acid is reacted directly with aappropriately functionalized surface to yield immobilized nucleic acid.Thus, for example, an iodoacetyl-modified surface (or otherthiol-reactive surface functionality) can react with a thiol-modifiednucleic acid to provide immobilized nucleic acids.

In accordance with the methods provided herein, the cross-linking agentis selected to provide a high density of nucleic acids immobilized onthe insoluble support. Without wishing to be bound by theory, it isbelieved that the high density of immobilized nucleic acids describedherein is due, at least in part, to a relatively rapid reactionoccurring between the cross-linking agent and the nucleic acid (e.g., athiol-modified nucleic acid), compared to other reactions previouslyused to immobilize nucleic acids. In addition, high density may at leastin part be due to a close spacing of the reactive groups (e.g., aminogroups of other reactive functionality) on the functionalized insolublesupport. Thus, reagents for modifying the surface will generally beselected to provide closely-spaced functionalities on the functionalizedsupport. The cross-linking agent (and other reagents used tofunctionalize the support surface or the nucleic acid molecule) can beselected to provide any desired spacing of the immobilized nucleic acidmolecules from the support surface, and to provide any desired spacingof the immobilized nucleic acids from each other. Thus, stericencumbrance of the nucleic acid molecules can be reduced or eliminatedby choice of an appropriate cross-linking agent. In certain embodiments,the cross-linking reagent can be selected to provide multiple reactivefunctionalities as used in dendrimer synthesis for attachment ofmultiple nucleic acids to a single cross-linking moiety. Preferably, thecross-linking agent is selected to be highly reactive with the nucleicacid molecule, to provide rapid, complete, and/or selective reaction. Inpreferred embodiments, the reaction volume of the reagents (e.g., thethiol group and the thiol-reactive functionality) is small.

Nucleic Acids and Linkers

Preferred nucleic acids for use herein are “thiol-modified nucleicacids,” i.e., nucleic acids derivatized to contain at least one reactivethiol moiety. As described in further detail in Example 1, below,nucleic acids containing at least one reactive thiol are preferably madeby treating a nucleic acid containing a 3′ or 5′ disulfide withakreducing agent, which preferably will not compete in subsequentreactions (i.e. will not react with an iodoacetimido functionality.Disulfide-derivatized nucleic acids can be synthesized according to avariety of methods. For example, a nucleic acid can be modified at the3′- or 5′-terminus by reaction with a disulfide-containing modifying areagent. Alternatively, a thiolated primer can by enzymatically ornon-enzymatically attached to the nucleic acid. A 5′-phosphoramidatefunctionality can also provide an attachment point for a thiol ordisulfide-containing y osine or deoxycytosine. Examples of reducingagents appropriate for reduction of a disulfide-modified nucleic acidinclude: tris-(2-carboxyethyl)phosphine (TCEP) (preferably aconcentration in the range of 1-100 mM (most preferably about 10 mM)) isreacted at a pH in the range of 3-6 (most preferably about 4.5), atemperature in the range of 20-45° C. (most preferably about 37° C.) fora time period in the range of about 1 to about 10 hrs (most preferablyfor about 5 hrs); dithiothreitol (preferably a concentration in therange of 25 to 100 mM (depending on whether the reactant is isolated) isreacted at a pH in the range of 6-10 (most preferably about 8) and at atemperature in the range of 25-45° C. (most preferably about 37° C.))for a time in the range of about 1 to about 10 hrs (most preferablyabout 5 hrs). TCE provides an advantage in the low pH at which it isreactive. This low pH effectively protonates thiols, thus suppressingnucleophilic reactions of thiols and resulting in fewer side reactionsthan with other disulfide reducing agents which are employed at higherpH.

As further described in Example 1, below, a preferred bifunctionalcross-linking agent is N-succinimidyl(4-iodacetyl) aminobenzoate (SIAB).Other crosslinking agents include, but are not limited to, dimaleimide,dithio-bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate(SATA), N-succinimidyl-3-(2-pyridyldithiol propionate (SPDP),succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) ad6-hydrazinonicotimide (HYNIC) may also be used in the novel process. Forfurther examples of cross-linking reagents, see, e.g., Wong “Chemistryof Protein Conjugation and Cross-Linking,” CRC Press (1991), andHermanson, “Bioconjugate Techniques” Academic Press (1995).

In preferred embodiments, the nucleic acid is immobilized using thephotocleavable linker moiety that is cleaved during mass spectrometry.Exemplary photolabile cross-linker include, but are not limited to,3-amino-(2-nitrophenyl)propionic acid (Brown et al. (1995) MolecularDiversity, pp.4-12 and Rothschild et. al. (1996) Nucleic Acids Res.24:361-66).

In a further embodiment of the methods of detecting alterations in atarget nucleic acid sequence provided herein and methods ofimmobilization, a single-stranded nucleic acid complementary to thetarget nucleic acid is immobilized to a surface through a linkage thatincludes a thiol group-thiol reactive functionality bond and acleavable, preferably a selectively cleavable, linker moiety.

Linkers

A target detection site can be directly linked to a solid support via areversible or irreversible bond between an appropriate functionality(L′) on the target nucleic acid molecule (T) and an appropriatefunctionality (L) on the capture molecule. A reversible linkage can besuch that it is cleaved under the conditions of mass spectrometry (i.e.,a photocleavable bond such as a charge transfer complex or a labile bondbeing formed between relatively stable organic radicals).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Biocone. Chem. 3:104-107),thereby releasing the targeted agent upon exposure to light.Photocleavable linkers that are cleaved upon exposure to light are known(see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp., 16th,Brunfeldt, K (Ed), pp. 105-110, which describes the use of a nitrobenzylgroup as a photocleavable protective group for cysteine; Yen et al.(1989) Makromol. Chem 190:69-82, which describes water solublephotocleavable copolymers, including hydroxypropylmethacrylamidecopolymer, glycine copolymer, fluorescein copolymer and methylrhodaminecopolymer; Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, whichdescribes a cross-linker and reagent that undergoes photolyticdegradation upon exposure to near UV light (350 nm); and Senter et al.(1985) Photochem. Photobiol 42:231-237, which describesnitrobenzyloxycarbonyl chloride cross linking reagents that producephotocleavable linkages), thereby releasing the targeted agent uponexposure to light. In preferred embodiments, the nucleic acid isimmobilized using the photocleavable linker moiety that is cleavedduring mass spectrometry.

Furthermore, the linkage can be formed with L′ being a quaternaryammonium group, in which case, preferably, the surface of the solidsupport carries negative charges which repel the negatively chargednucleic acid backbone and thus facilitate the desorption required foranalysis by a mass spectrometer. Desorption can occur either by the heatcreated by the laser pulse and/or, depending on L,′ by specificabsorption of laser energy which is in resonance with the L′chromophore.

Thus, the L-L′ chemistry can be of a type of disulfide bond (chemicallycleavable, for example, by mercaptoethanol or dithioerythrol), abiotin/streptavidin system, a heterobifunctional derivative of a tritylether group (see, e.g., Köster et al. (1990) “A Versatile Acid-LabileLinker for Modification of Synthetic Biomolecules,” Tetrahedron Letters31:7095) that can be cleaved under mildly acidic conditions as well asunder conditions of mass spectrometry, a levulinyl group cleavable underalmost neutral conditions with a hydrazinium/acetate buffer, anarginine-arginine or lysine-lysine bond cleavable by an endopeptidaseenzyme like trypsin or a pyrophosphate bond cleavable by apyrophosphatase, or a ribonucleotide bond in between theoligodeoxynucleotide sequence, which can be cleaved, for example, by aribonuclease or alkali.

The functionalities, L and L,′ can also form a charge transfer complexand thereby form the temporary L-L′ linkage. Since in many cases the“charge-transfer band” can be determined by UV/vis spectrometry (see,e.g., Organic Charge Transfer Complexes by R. Foster, Academic Press,1969), the laser energy can be tuned to the corresponding energy of thecharge-transfer wavelength and, thus, a specific desorption off thesolid support can be initiated. Those skilled in the art will recognizethat several combinations can serve this purpose and that the donorfunctionality can be either on the solid support or coupled to thenucleic acid molecule to be detected or vice versa.

In yet another approach, a reversible L-L′ linkage can be generated byhomolytically forming relatively stable radicals. Under the influence ofthe laser pulse, desorption (as discussed above) as well as ionizationwill take place at the radical position. Those skilled in the art willrecognize that other organic radicals can be selected and that, inrelation to the dissociation energies needed to homolytically cleave thebond between them, a corresponding laser wavelength can be selected (seee.g., Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).

When performing exonuclease sequencing using MALDI-TOF MS, a singlestranded DNA molecule immobilized via its 5′-end to a solid support isunilaterally degraded with a 3′-processive exonuclease and the molecularweight of the degraded nucleotide is determined sequentially. ReverseSanger sequencing reveals the nucleotide sequence of the immobilizedDNA. By adding a selectively cleavable linker, not only can the mass ofthe free nucleotides be determined but also, upon removal of thenucleotides by washing, the mass of the remaining fragment can bedetected by MALDI-TOF upon cleaving the DNA from the solid support.Using selectively cleavable linkers, such as the photocleavable andchemical cleavable linkers provided herein, this cleavage can beselected to occur during the ionization and volatizing steps ofMALDI-TOF. The same rationale applies for a 5′ immobilized strand of adouble stranded DNA that is degraded while in a duplex. Likewise, thisalso applies when using a 5′-processive exonuclease and the DNA isimmobilized through the 3′-end to the solid support.

As noted, at least three version of immobilization are contemplatedherein: 1) the target nucleic acid is amplified or obtained (the targetsequence or surrounding DNA sequence must be known to make primers toamplify or isolated); 2) the primer nucleic acid is immobilized to thesolid support and the target nucleic acid is hybridized thereto (this isfor detecting the presence of or sequencing a target sequence in asample); or 3) a double stranded DNA (amplified or isolated) isimmobilized through linkage to one predetermined strand; the DNA isdenatured to eliminate the duplex and then a high concentration of acomplementary primer or DNA with identity upstream from the target siteis added and a strand displacement occurs and the primer is hybridizedto the immobilized strand.

In the embodiments where the primer nucleic acid is immobilized on thesolid support and the target nucleic acid is hybridized thereto, theinclusion of the cleavable linker allows the primer DNA to beimmobilized at the 5′-end so that free 3′-OH is available for nucleicacid synthesis (extension) and the sequence of the “hybridized” targetDNA can be determined because the hybridized template can be removed bydenaturation and the extended DNA products cleaved from the solidsupport for MALDI-TOF MS. Similarly for 3), the immobilized DNA strandcan be elongated when hybridized to the template and cleaved from thesupport. Thus, Sanger sequencing and primer oligo base extension(PROBE), discussed below, extension reactions can be performed using animmobilized primer of a known, upstream DNA sequence complementary to aninvariable region of a target sequence. The nucleic acid from the personis obtained and the DNA sequence of a variable region (deletion,insertion, missense mutation that cause genetic predisposition ordiseases, or the presence of viral/bacterial or fungal DNA) not only isdetected, but the actual sequence and position of the mutation is alsodetermined.

In other cases, the target DNA must be immobilized and the primerannealed. This requires amplifying a larger DNA based on known sequenceand then sequencing the immobilized fragments (i.e., the extendedfragments are hybridized but not immobilized to the support as describedabove). In these cases, it is not desirable to include a linker becausethe MALDI-TOF spectrum is of the hybridized DNA; it is not necessary tocleave the immobilized template.

Any linker known to those of skill in the art for immobilizing nucleicacids to solid supports may be used herein to link the nucleic acid to asolid support. The preferred linkers herein are the selectivelycleavable linkers, particularly those exemplified herein. Other linkersinclude, acid cleavable linkers, such as bismaleimideothoxy propane,acid-labile trityl linkers.

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thetargeted agent to permit it to be more readily accessible to reaction.Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584-589) and acidlabile transfering conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e., Welhöner et al. (1991) J. Biol. Chem. 266:4309-4314).

Photocleavable Linkers

Photocleavable linkers are provided. In particular, photocleavablelinkers as their phosphoramidite derivatives are provided for use insolid phase synthesis of oligonucleotides. The linkers containo-nitrobenzyl moieties and phosphate linkages which allow for completephotolytic cleavage of the conjugates within minutes upon UVirradiation. The UV wavelengths used are selected that the irradiationwill not damage the oligonucleotides and are preferably about 350-380nm, more preferably 365 nm. The photocleavable linkers provided hereinpossess comparable coupling efficiency as compared to commonly usedphosphoramidite monomers (see, Sinha et al. (1983) Tetrahedron Lett.24:5843-5846; Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557;Beaucage et al. (1993) Tetrahedron 49:6123-6194; and Matteucci et al.(1981) J. Am. Chem. Soc. 103:3185-3191).

In one embodiment, the photocleavable linkers have formula I:

where R²⁰ is w-(4,4′-dimethoxytrityloxy)alkyl or w-hydroxyalkyl; R²¹ isselected from hydrogen, alkyl, aryl, alkoxycarbonyl, aryloxycarbonyl andcarboxy; R²² is hydrogen or (dialkylamino)(w-cyanoalkoxy)P-; t is 0-3;and R⁵⁰ is alkyl, alkoxy, aryl or aryloxy.

In a preferred embodiment, the photocleavable linkers have formula II:

where R²⁰ is w-(4,4′-dimethoxytrityloxy)alkyl, w-hydroxyalkyl or alkyl;R²¹ is selected from hydrogen, alkyl, aryl, alkoxycarbonyl,aryloxycarbonyl and carboxy; R²² is hydrogen or(dialkylamino)(w-cyanoalkoxy)P-; and X²⁰ is hydrogen, alkyl or OR²⁰.

In particularly preferred embodiments, R²⁰ is3-(4,4′-dimethoxytrityloxy)propyl, 3-hydroxypropyl or methyl; R²¹ isselected from hydrogen, methyl and carboxy; R²² is hydrogen or(diisopropylamino)(2-cyanoethoxy)P-; and X²⁰ is hydrogen, methyl orOR²⁰. In a more preferred embodiment, R²⁰ is3-(4,4′-dimethoxytrityloxy)propyl; R²¹ is methyl; R²² is(diisopropylamino)(2-cyanoethoxy)P-; and X²⁰ is hydrogen. In anothermore preferred embodiment, R²⁰ is methyl; R²¹ is methyl; R²² is(diisopropylamino)(2-cyanoethoxy)P-; and X²⁰ is3-(4,4′-dimethoxytrityloxy)propoxy.

In another embodiment, the photocleavable linkers have formula III

where R²³ is hydrogen or (dialkylamino)(w-cyanoalkoxy)P-; and R²⁴ isselected from w-hydroxyalkoxy, w-(4,4′-dimethoxytrityloxy)alkoxy,w-hydroxyalkyl and w-(4,4′-dimethoxytrityloxy)alkyl, and isunsubstituted or substituted on the alkyl or alkoxy chain with one ormore alkyl groups; r and s are each independently 0-4; and R⁵⁰ is alkyl,alkoxy, aryl or aryloxy. In certain embodiments, R²⁴ is w-hydroxyalkylor w-(4,4′-dimethoxytrityloxy)alkyl, and is substituted on the alkylchain with a methyl group.

In preferred embodiments, R²³ is hydrogen or(diisopropylamino)(2-cyanoethoxy)P-; and R²⁴ is selected from3-hydroxypropoxy, 3-(4,4′-dimethoxytrityloxy)propoxy, 4-hydroxybutyl,3-hydroxy-1-propyl, 1-hydroxy-2-propyl, 3-hydroxy-2-methyl-1-propyl,2-hydroxyethyl, hydroxymethyl, 4-(4,4′-dimethoxytrityloxy)buty),3-(4,4′-dimethoxytrityloxy)-1-propyl, 2-(4,4′-dimethoxytrityloxy)ethyl,1-(4,4′-dimethoxytrityloxy)-2-propyl,3-(4,4′-dimethoxytriyioxy)-2-methyl-1-propyl and4,4′-dimethyoxytrityloxymethyl.

In more preferred embodiments, R²³ is(diisopropylamino)(2-cyanoethoxy)P-; r and s are 0; and R²⁴ is selectedfrom 3-(4,4′-dimethoxytrityloxy)propoxy,4-(4,4′-dimethoxytrityloxy)butyl, 3-(4,4′-dimethoxytrityloxy)propyl,2-(4,4′-dimethoxytrityloxy)ethyl, 1-(4,4′-dimethoxytrityloxy)-2-propyl,3-(4,4′-dimethoxytriyloxy)-2-methyl-1-propyl and4,4′-dimethyoxytrityloxymethyl. R²⁴ is most preferably3-(4,4′-dimethoxytrityloxy)propoxy.

Preparation of the Photocleavable Linkers

A. Preparation of Photocleavable Linkers of Formulae I or II

Photocleavable linkers of formulae I or II may be prepared by themethods described below, by minor modification of the methods bychoosing the appropriate starting materials or by any other methodsknown to those of skill in the art.

In the photocleavable linkers of formula it where X²⁰ is hydrogen, thelinkers may be prepared in the following manner. Alkylation of5-hydroxy-2-nitrobenzaldehyde with an w-hydroxyalkyl halide, e.g.,3-hydroxypropyl bromide, followed by protection of the resulting alcoholas, e.g., a silyl ether, provides a5-(w-silyloxyalkoxy)-2-nitrobenzaldehyde. Addition of an organometallicto the aldehyde affords a benzylic alcohol. Organometallics which may beused include trialkylaluminums (for linkers where R²¹ is alkyl), such astrimethylaluminum, borohydrides (for linkers where R²¹ is hydrogen),such as sodium borohydride, or metal cyanides (for linkers where R²¹ iscarboxy or alkoxycarbonyl), such as potassium cyanide. In the case ofthe metal cyanides, the product of the reaction, a cyanohydrin, wouldthen be hydrolyzed under either acidic or basic conditions in thepresence of either water or an alcohol to afford the compounds ofinterest.

The silyl group of the side chain of the resulting benzylic alcohols maythen be exchanged for a 4,4′-dimethoxytriyl group by desilylation with,e.g., tetrabutylammonium fluoride, to give the corresponding alcohol,followed by reaction with 4,4′-dimethoxytrityl chloride. Reaction with,e.g., 2-cyanoethyl diisopropylchlorophosphoramidite affords the linkerswhere R²² is (dialkylamino)(w-cyanoalkoxy)P-.

A specific example of a synthesis of a photocleavable linker of formulaII is shown in the following scheme, which also demonstrates use of thelinker in oligonucleotide synthesis. This scheme is intended to beillustrative only and in no way limits the scope of the invention.Experimental details of these synthetic transformations are provided inthe Examples.

Synthesis of the linkers of formula II where X²⁰ is OR²⁰,3,4-dihydroxyacetophenone is protected selectively at the 4-hydroxyl byreaction with, e.g., potassium carbonate and a silyl chloride. Benzoateesters, propiophenones, butyrophenones, etc. may be used in place of theacetophenone. The resulting 4-silyloxy-3-hydroxyacetophenone is thenalkylated at the with an alkyl halide (for linkers where R²⁰ is alkyl)at the 3-hydroxyl and desilylated with, e., tetrabutylammonium fluorideto afford a 3-alkoxy-4-hydroxyacetophenone. This compound is thenalkylated at the 4-hydroxyl by reaction with an w-hydroxyalkyl halide,e.g., 3-hydroxypropyl bromide, to give a4-(w-hydroxyalkoxy)-3-alkoxyacetophenone. The side chain alcohol is thenprotected as an ester, e.g., an acetate. This compound is then nitratedat the 5-position with, e.g., concentrated nitric acid to provide thecorresponding 2-nitroacetophenones. Saponification of the side chainester with, e.g., potassium carbonate, and reduction of the ketone with,e.g., sodium borohydride, in either order gives a2-nitro-4-(w-hydroxyalkoxy)-5-alkoxybenzylic alcohol.

Selective protection of the side chain alcohol as the corresponding4,4′-dimethoxytrityl ether is then accomplished by reaction with4,4′-dimethoxytrityl chloride. Further reaction with, e.g., 2-cyanoethyldiisopropylchlorophosphoramidite affords the linkers where R²² is(dialkylamino)(w-cyanoalkoxy)P-.

A specific example of the synthesis of a photocleavable linker offormula II is shown the following scheme. This scheme is intended to beillustrative only and in no way limit the scope of the invention.Detailed experimental procedures for the transformations shown are foundin the Examples.

B. Preparation of Photocleavable Linkers of Formula III

Photocleavable linkers of formula III may be prepared by the methodsdescribed below, by minor modification of the methods by choosingappropriate starting materials, or by other methods known to those ofskill in the art.

In general, photocleavable linkers of formula III are prepared fromw-hydroxyalkyl- or alkoxyaryl compounds, in particular w-hydroxy-alkylor alkoxy-benzenes. These compounds are commercially available, or maybe prepared from an w-hydroxyalkyl halide (e.g., 3-hydroxypropylbromide) and either phenyllithium (for the w-hydroxyalkylbenzenes) orphenol (for the w-hydroxyalkoxybenzenes). Acylation of the w-hydroxylgroup (e.g., as an acetate ester) followed by Friedel-Crafts acylationof the aromatic ring with 2-nitrobenzoyl chloride provides a4-(w-acetoxy-alkyl or alkoxy)-2-nitrobenzophenone. Reduction of theketone with, e.g., sodium borohydride, and saponification of the sidechain ester are performed in either order to afford a2-nitrophenyl-4-(hydroxy-alkyl or alkoxy)phenylmethanol. Protection ofthe terminal hydroxyl group as the corresponding 4,4′-dimethoxytritylether is achieved by reaction with 4,4′-dimethoxytrityl chloride. Thebenzylic hydroxyl group is then reacted with, e.g., 2-cyanoethyldiisopropylchlorophosphoramidite to afford linkers of formula II whereR²³ is (dialkylamino)(w-cyanoalkoxy)P-. Other photocleavable linkers offormula III may be prepared by substituting 2-phenyl-1-propanol or2-phenylmethyl-1-propanol for the w-hydroxy-alkyl or alkoxy-benzenes inthe above synthesis. These compounds are commercially available, but mayalso be prepared by reaction of, e.g., phenylmagnesium bromide orbenzylmagnesium bromide, with the requisite oxirane (i.e., propyleneoxide) in the presence of catalytic cuprous ion.

Chemically Cleavable Linkers

A variety of chemically cleavable linkers may be used to introduce acleavable bond between the immobilized nucleic acid and the solidsupport. Acid-labile linkers are presently preferred chemicallycleavable linkers for mass spectrometry, especially MALDI-TOF MS,because the acid labile bond is cleaved during conditioning of thenucleic acid upon addition of the 3-HPA matrix solution. The acid labilebond can be introduced as a separate linker group, e.g., the acid labiletrityl groups or may be incorporated in a synthetic nucleic acid linkerby introducing one or more silyl internucleoside bridges usingdiisopropylsilyl, thereby forming diisopropylsilyl-linkedoligonucleotide analogs. The diisopropylsilyl bridge replaces thephoshodiester bond in the DNA backbone and under mildly acidicconditions, such as 1.5% trifluoroacetic acid (TFA) or 3-HPA/1% TFAMALDI-TOF matrix solution, results in the introduction of one or moreintra-strand breaks in the DNA molecule. Methods for the preparation ofdiisopropylsilyl-linked oligonucleotide precursors and analogs are knownto those of skill in the art (see e.g., Saha et al. (1993) J. Org. Chem.58:7827-7831). These oligonucleotide analogs may be readily preparedusing solid state oligonucleotide synthesis methods usingdiisopropylsilyl derivatized deoxyribonucleosides.

Mass Modification of Nucleic Acids

In certain embodiments, nucleic acids modified at positions other thanthe 3′- or 5′-terminus can be used. Modification of the sugar moiety ofa nucleotide at positions other than the 3′ and 5′ position is possiblethrough conventional methods. Also, nucleic acid bases can be modified,e.g., by modification of C-5 of dT with a linker arm, e.g., as describedin F. Eckstein, ed., “Oligonucleotides and Analogues: A PracticalApproach,” IRL Press (1991). Such a linker arm can be modified toinclude a thiol moiety. Alternatively, backbone-modified nucleic acids(e.g., phosoroamidate DNA) can be used so that the thiol group can beattached to the nitrogen center provided by the modified phosphatebackbone.

In preferred embodiments, modification of a nucleic acid, e.g., asdescribed above, does not substantially impair the ability of thenucleic acid or nucleic sequence to hybridize to its complement. Thus,any modification should preferably avoid substantially modifying thefunctionalities of the nucleic acid which are responsible forWatson-Crick base pairing. The nucleic acid can be modified such that anon-terminal thiol group is present, and the nucleic acid, whenimmobilized to the support, is capable of self-complementary basepairing to form a “hairpin” structure having a duplex region.

Solid Supports and Substrates

Examples of insoluble supports and substrates for use herein include,but are not limited to, beads (silica gel, controlled pore glass,magnetic beads, Sephadex/Sepharose beads, cellulose beads, etc.),capillaries, flat supports such as glass fiber filters, glass surfaces,metal surfaces (steel, gold, silver, aluminum, silicon and copper),plastic materials including multiwell plates or membranes (e.g., ofpolyethylene, polypropylene, polyamide, polyvinyldenedifluoride),wafers, combs, pins (e.g., arrays of pins suitable for combinatorialsynthesis or analysis) or beads in pits of flat surfaces such as wafers(e.g., silicon wafers), with or without filter plates.

Mass Spectrometry

Once immobilized, the nucleic acids can be analyzed by any of a varietyof means including, for example, spectrometric techniques such asUV/VIS, IR, fluorescence, chemiluminescence, or NMR spectroscopy, massspectrometry, or other methods know in the art, or combinations thereof.Preferred mass spectrometer formats include the ionization (I)techniques, such as matrix assisted laser desorption (MALDI), continuousor pulsed electrospray (ESI) and related methods (e.g. lonspray orThermospray), or massive cluster impact (MCI); these ion sources can bematched with detection formats including linear or reflectrontime-of-flight (TOF), single or multiple quadruple, single or multiplemagnetic sector, Fourier Transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof to yield a hybrid detector (e.g.,ion-trap/time-of-flight). For ionization, numerous matrix/wavelengthcombinations (MALDI) or solvent combinations (ESI) can be employed.

Preparation of DNA Arrays

Methods and systems for preparing arrays of sample material for analysisby a diagnostic tool are provided herein. For example, FIG. 1illustrates one system for preparing arrays of sample material foranalysis by a diagnostic tool. FIG. 1 depicts a system 10 that includesa data processor 12, a motion controller 14, a robotic arm assembly 16,a monitor element 18A, a central processing unit 18B, a microliter plateof source material 20, a stage housing 22, a robotic arm 24, a stage 26,a pressure controller 28, a conduit 30, a mounting assembly 32, a pinassembly 38, and substrate elements 34. In the view shown by FIG. 1, itis also illustrated that the robotic assembly 16 can include a moveablemount element 40 and a horizontal slide groove 42. The robotic arm 24can optionally pivot about a pin 36 to increase the travel range of thearm 24 so that arm 24 can disposes the pin assembly 38 above the sourceplate 20.

The data processor 12 depicted in FIG. 1 can be a conventional digitaldata processing system such as an IBM PC compatible computer system thatis suitable for processing data and for executing program instructionsthat will provide information for controlling the movement and operationof the robotic assembly 16. It will be apparent to one skilled in theart that the data processor unit 12 can be any type of system suitablefor processing a program of instructions signals that will operate therobotic assembly that is integrated into the robotic housing 16.Optionally the data processor 12 can be a micro-controlled assembly thatis integrated into robotic housing 16. In further alternativeembodiments, the system 10 need not be programmable and can be asingleboard computer having a firmware memory for storing instructionsfor operating the robotic assembly 16.

In the embodiment depicted in FIG. 1, there is a controller 14 thatelectronically couples between the data processor 12 and the roboticassembly 16. The depicted controller 14 is a motion controller thatdrives the motor elements of the robotic assembly 16 for positioning therobotic arm 24 at a selected location. Additionally, the controller 14can provide instructions to the robotic assembly 16 to direct thepressure controller 28 to control the volume of fluid ejected from theindividual pin elements of the depicted pin assembly 38. The design andconstruction of the depicted motion controller 14 follows fromprinciples well known in the art of electrical engineering, and anycontroller element suitable for driving the robotic assembly 16 can bepracticed without departing from the scope thereof.

The robotic assembly 16 depicted in FIG. 1 electronically couples to thecontroller 14. The depicted robotic assembly 16 is a gantry system thatincludes an XY table for moving the robotic arm about a XY plane, andfurther includes a Z axis actuator for moving the robotic armorthogonally to that XY plane. The robotic assembly 16 depicted in FIG.1 includes an arm 24 that mounts to the XY stage which moves the armwithin a plane defined by the XY access. In the depicted embodiment, theXY table is mounted to the Z actuator to move the entire table along theZ axis orthogonal to the XY plane. In this way, the robotic assemblyprovides three degrees of freedom that allows the pin assembly 38 to bedisposed to any location above the substrates 34 and the source plate 20which are shown in FIG. 1 as sitting on the stage 26 mounted to therobotic assembly 16.

The depicted robotic assembly 16 follows from principles well known inthe art of electrical engineering and is just one example of a roboticassembly suitable for moving a pin assembly to locations adjacent asubstrate and source plate such as the depicted substrate 34.Accordingly, it will be apparent to one of ordinary skill in the artthat alternative robotic systems can be practiced following thedescriptions herein without departing from the scope thereof.

FIG. 1 depicts an embodiment of a robotic assembly 16 that includes apressure controller 28 that connects via a conduit 30 to the mount 32that connects to the pin assembly 38. In this embodiment the mount 32has an interior channel for fluidicly coupling the conduit 30 to the pinassembly 38. Accordingly, the pressure controller 28 is fluidiclycoupled by the conduit 30 and the mount 32 to the pin assembly 38. Inthis way the controller 14 can send signals to the pressure controller28 to control selectively a fluid pressure delivered to the pin assembly38.

FIG. 2 depicts one embodiment of a pin assembly 50 suitable for practicewith the system depicted in FIG. 1 which includes the pressurecontroller 28. In the depicted embodiment, the pin assembly 50 includesa housing formed from an portion 52 and a lower portion 54 that arejoined together by the screws 56A and 56B to define an interior chambervolume 58. FIG. 2 further depicts that to fluidicly seal the interiorchamber volume 58 the housing can include a seal element depicted inFIG. 2 as an O-ring gasket 60 that sits between the upper block and thelower block 54 and surrounds completely the perimeter of the interiorchamber volume 58. FIG. 2 further depicts that the pin assembly 50includes a plurality of vesicles 62A-62D, each of which include an axialbore extending therethrough to form the depicted holding chambers64A-64D. Each of the depicted vesicles extends through a respectiveaperture 68A-68D disposed within the lower block 54 of the housing.

As further shown in the depicted embodiment, each of the vesicles62A-62D has an upper flange portion that sits against a seal element70A-70D to form a fluid-tight seal between the vesicle and the lowerblock 54 to prevent fluid from passing through the apertures 68A-68D. Tokeep the seal tight, the depicted pin assembly 50 further includes a setof biasing elements 74A-74D depicted in FIG. 2 as springs which, in thedepicted embodiments, are in a compressed state to force the flangeelement of the vesicles 62A-62D against their respective seal elements70A-70D. As shown in FIG. 2, the biasing elements 74A-74D extend betweenthe vesicles and the upper block 52. Each of the springs 74A-74D can befixedly mounted to a mounting pad 76A-76D where the spring elements canattach to the upper block 52. The upper block 52 further includes anaperture 78 depicted in FIG. 2 as a centrally disposed aperture thatincludes a threaded bore for receiving a swagelok 80 that can berotatably mounted within the aperture 78.

As further depicted in FIG. 2, the swagelok 80 attaches by a conduit toa valve 82 than can connect the swagelok 80 to a conduit 84 that can becoupled to a pressure source, or alternatively can couple the swagelok80 to a conduit 86 that provides for venting of the interior chamber 58.A central bore 88 extends through the swagelok 80 and couples to thetubing element which further connects to the valve 82 to therebyfluidicly and selectively couple the interior chamber volume 58 toeither a pressure source, or a venting outlet.

The pin assembly 50 described above and depicted in FIG. 2 disposedabove a substrate element 90 that includes a plurality of wells 92 thatare etched into the upper surface of the substrate 90. As illustrated byFIG. 2, the pitch of the vesicles 62A-62D is such that each vesicle isspaced from the adjacent vesicles by a distance that is an integralmultiple of the pitch distance between wells 92 etched into the uppersurface of the substrate 90. As will be seen from the followingdescription, this spacing facilitates the parallel dispensing of fluid,such that fluid can be dispensed into a plurality of wells in a singleoperation. Each of the vesicles can be made from stainless steel,silica, polymeric material or any other material suitable for holdingfluid sample. In one example, 16 vesicles are employed in the assembly,which are made of hardened beryllium copper, gold plated over nickelplate. They are 43.2 mm long and the shaft of the vesicle is graduatedto 0.46 mm outer diameter with a concave tip. Such a pin was chosensince the pointing accuracy can be approximately 501 micrometers.However, it will be apparent that any suitable pin style can be employedfor the device, including but not limited to flat, star-shaped, concave,pointed solid, pointed semi-hollow, angled on one or both sides, orother such geometries.

FIG. 3 shows from a side perspective the lower block 54 of the pinassembly 50 depicted in FIG. 2. FIG. 3 shows approximate dimensions forone pin assembly. As shown, the lower block 54 has a bottom plate 98 anda surrounding shoulder 100. The bottom plate 98 is approximately 3 mm inthickness and the shoulder 100 is approximately 5 mm in thickness.

FIG. 4 shows from an overhead perspective the general structure anddimensions for one lower block 54 suitable for use with the pin assemblyfor use with the pin assembly 50 shown in FIG. 2. As shown in FIG. 4,the lower block 54 includes a four-by-four matrix of apertures 68 toprovide 16 apertures each suitable for receiving a vesicle. As describedabove with reference to FIG. 2, the spacing between the aperture 68 istypically an integral multiple of the distance between wells on asubstrate surface as well as the wells of a source plate. Accordingly, apin assembly having the lower block 54 as depicted in FIG. 4 candispense fluid in up to 16 wells simultaneously. FIG. 4 also showsgeneral dimensions of one lower block 54 such that each side of block 54is generally 22 mm in length and the pitch between aperture 68 isapproximately 4.5 mm. Such a pitch is suitable for use with a substratewhere fluid is to be dispensed at locations approximately 500 μm apart,as exemplified by the substrate 90 of FIG. 2. FIG. 4 also shows that thelower block 54 can include an optional O-ring groove 94 adapted forreceiving an O-ring seal element, such as the seal element 60 depictedin FIG. 2. It is understood that such a groove element 94 can enhanceand improve the fluid seal formed by the seal element 60.

The pinblock can be manufactured of stainless steel as this material canbe drilled accurately to about +25 μm, but a variety of probe materialscan also be used, such as G10 laminate, PMMA or other suitable material.The pin block can contain any number of apertures and is shown with 16receptacles which hold the 16 pins in place. To increase the pointingaccuracy of each pin, an optional alignment place can be placed belowthe block so that about 6 mm of the pin tip is left exposed to enabledipping into the wells of a microtiter plate. The layout of the probesin the depicted tool is designed to coordinate with a 384-wellmicrotiter plate, thus the center-to-center spacing of the probes in 4.5mm. An array of 4×4 probes was chosen since it would produce an arraythat would fit in less than one square inch, which is the travel rangeof an xy stage of a MALDI TOF MS employed by the assignee. The pintoolassembly is completed with a stainless steel cover on the top side ofthe device which is then attached onto the Z-arm of the robot.

With references to FIG. 1, the robotic assembly 16 employs a pin toolassembly 38 that is configured similarly as the pin tool assembly 50depicted in FIG. 2. The pressure controller 28 selectively controls thepressure within chamber 58. With this embodiment, a control programoperates on the data processor 12 to control the robotic assembly 16 ina way that the assembly 16 prints an array of elements on the substrates34.

In a first step, FIG. 5A, the program can direct the robotic assembly 16to move the pin assembly 38 to be disposed above the source plate 20.The robotic assembly 16 will then dip the pin assembly into the sourceplate 20 which can be a 384 well DNA source plate. As shown in FIG. 4the pin assembly can include 16 different pins such that the pinassembly 50 will dip 16 pins into different 16 wells of the 384 well DNAsource plate 20. Next the data processor 12 will direct the motioncontroller 14 to operate the robotic assembly 16 to move the pinassembly to a position above the surface of the substrate 34. Thesubstrate 34 can be any substrate suitable for receiving a sample ofmaterial and can be formed of silicon, plastic, metal, or any other suchsuitable material. Optionally the substrate will have a flat surface,but can alternatively include a pitted surface, a surface etched withwells or any other suitable surface typography. The program operating ondata processor 12 can then direct the robotic assembly, through themotion controller 14, to direct the pressure controller 28 to generate apositive pressure within the interior chamber volume 58. In thispractice, the positive interior pressure will force fluid from theholding chambers of vesicles 62 to eject fluid from the vesicles andinto a respective well 92 of the substrate 90.

The program operating on data processor 12 can also direct thecontroller 14 to control the pressure controller 28 to control fillingthe holding chambers with source material from the source plate 20. Thepressure controller 28 can generate a negative pressure within theinterior chamber volume 58 of the pin assembly. This will cause fluid tobe drawn up into the holding chambers of the vesicles 62A-62D. Thepressure controller 28 can regulate the pressure either by open-loop orclosed-loop control to avoid having fluid overdrawn through the holdingchambers and spilled into the interior chamber volume 58. Loop controlsystems for controlling pressure are well known in the art and anysuitable controller can be employed. Such spillage could causecross-contamination, particularly if the source material drawn from thesource plate 20 varies from well to well.

In an alternative practice of the invention, each of the holdingchambers 64A-64D is sufficiently small to allow the chambers to befilled by capillary action. In such a practice, the pin assembly canconsist of an array of narrow bore needles, such as stainless steelneedles, that extend through the apertures of the lower block 54. Theneedles that are dipped into source solutions will be filled bycapillary action. In one practice, the length of capillary which is tobe filled at atmospheric pressure is determined approximately by:$H = \frac{2y}{PGR}$

where H equals Height, gamma equals surface tension, P equals solutiondensity, G equals gravitational force and R equals needle radius. Thusthe volume of fluid held by each vesicle can be controlled by selectingthe dimensions of the interior bore. It is understood that at roomtemperature water will fill a 15 cm length of 100 μm radius capillary.Thus, a short bore nanoliter volume needle will fill to full capacity,but should not overflow because the capillary force is understood to betoo small to form a meniscus at the top of the needle orifice. Thisprevents cross-contamination due to spillage. In one embodiment, thevesicles of the pin assembly can be provided with different sizedinterior chambers for holding and dispensing different volumes of fluid.

In an alternative practice, to decrease the volume of liquid that isdrawn into the holding chambers of the vesicles, a small positivepressure can be provided within the interior chamber volume 58 by thepressure controller 28. The downward force created by the positivepressure can be used to counter the upward capillary force. In this way,the volume of fluid that is drawn by capillary force into the holdingchambers of the vesicles can be controlled.

FIG. 5B shows that fluid within the holding chambers of the needle canbe dispensed by a small positive pressure introduced through the centralbore 88 extending through a swagelok 80. By regulating the pressurepulse that is introduced into the interior chamber volume 58, fluid canbe ejected either as a spray or by droplet formation at the needle tip.It is understood that the rate of dispensing, droplet versus spray,depends in part upon the pressure applied by the pressure controller 28.In one practice, pressure is applied in the range of between 10 and1,000 Torr of atmospheric pressure.

To this end the data processor 12 can run a computer program thatcontrols and regulates the volume of fluid dispensed. The program candirect the controller 28 to eject a defined volume of fluid, either bygenerating a spray or by forming a drop that sits at the end of thevesicle, and can be contacted with the substrate surface for dispensingthe fluid thereto.

FIGS. 5C and 5D show the earlier steps shown in FIGS. 5A-5B can again beperformed, this time at a position on the substrate surface that isoffset from the earlier position. In the depicted process, the pin toolis offset by a distance equal to the distance between two wells 92. Itwill be apparent that other offset printing techniques can be employedwithout departing from the scope of the invention.

It will be understood that several advantages of the pin assemblydepicted in FIG. 2 are achieved. For example, rinsing between dispensingevents is straightforward, requiring only single or multiple pinfillings and emptying events with a rinse solution. Moreover, since allholding chambers fill to full capacity, the accuracy of the volumesdispensed varies only according to needle inner dimensions which can becarefully controlled during pin production. Further the device is costeffective, with the greatest expense attributed to the needles, howeverbecause no contact with a surface is required, the needles are exposedto little physical strain or stress, making replacement rare andproviding long life.

Alternatively, deposition of sample material onto substrate surface caninclude techniques that employ pin tool assemblies that have solid pinelements extending from a block wherein a robotic assembly dips thesolid pin elements of the pin assembly into a source of sample materialto wet the distal ends of the pins with the sample materials.Subsequently the robotic assembly can move the pin assembly to alocation above the substrate and then lower the pin assembly against thesurface of the substrate to contact the individual wetted pins againstthe surface for spotting material of the substrate surface.

FIGS. 6A and 6B depict another alternative system for dispensingmaterial on or to the surface of the substrate. In particular, FIG. 6Adepicts a jet printing device 110 which includes a capillary element112, a transducer element 114 and orifice (not shown) 118, a fluidconduit 122, and a mount 124 connecting to a robotic arm assembly, suchas the robotic arm 24 depicted in FIG. 1. As further shown in FIG. 6Athe jet assembly 110 is suitable for ejecting from the orifice 118 aseries of drops 120 of a sample material for dispensing sample materialonto the surface 128.

The capillary 112 of the jet assembly 110 can be a glass capillary, aplastic capillary, or any other suitable housing that can carry a fluidsample and that will allow the fluid sample to be ejected by the actionof a transducer element, such as the transducer element 114. Thetransducer element 114 depicted in FIG. 6A is a piezo electrictransducer element which forms around the parameter of the capillary 112and can transform an electrical pulse received from the pulse generatorwithin a robotic assembly 16 to cause fluid to eject from the orifice118 of the capillary 112. One such jet assembly having a piezoelectrictransducer element is manufactured by MicroFab Technology, Inc., ofGermany. Any jet assembly, however, that is suitable for dispensingdefined and controlled the volumes of fluid can be used herein includingthose that use piezoelectric transducers, electric transducers,electrorestrictive transducers, magnetorestrictive transducers,electromechanical transducers, or any other suitable transducer element.In the depicted embodiment, the capillary 112 has a fluid conduit 122for receiving fluid material. In an optional embodiment, fluid can bedrawn into the capillary by action of a vacuum pressure that will drawfluid through the orifice 118 when the orifice 118 is submerged in asource of fluid material. Other embodiments of the jet assembly 110 canbe practiced with the invention without departing from the scopethereof.

FIG. 6B illustrates a further alternative assembly suitable for p beingcarried on the robotic arm of a robotic assembly, such as the assembly16 depicted in FIG. 1. FIG. 6B illustrates four jet assemblies connectedtogether, 130A-130D. Similar to the pin assembly in FIG. 2, the jetassembly depicted in FIG. 6B can be employed for the parallel dispensingof fluid material. It will be obvious to one of ordinary skill in theart of electrical engineering, that each of the jet assemblies 130A-130Dcan be operated independently of the others, for allowing the selectivedispensing of fluid from select ones of the jet assemblies. Moreover,each of the jet assemblies 130A-130D can be independently controlled toselect the volume of fluid that is dispensed from each respected one ofthe assembly 130A-130D. Other modifications and alterations can be madeto the assembly depicted in FIG. 6B without departing from the scope ofthe invention.

Methods for rapidly analyzing sample materials are also provided. Tothis end sample arrays can be formed on a substrate surface according toany of the techniques discussed above. The sample arrays are thenanalyzed by mass spectrometry to collect spectra data that isrepresentative of the composition of the samples in the array. It isunderstood that the above methods provide processes that allow forrapidly dispensing definite and controlled volumes of analyte material.In particular these processes allow for dispensing sub to low nanolitervolumes of fluid. These low volume deposition techniques generate samplearrays well suited for analysis by mass spectrometry. For example, thelow volumes yield reproducibility of spot characteristics, such asevaporation rates and reduced dependence on atmospheric conditions suchas ambient temperature and light

Continuing with the example shown in FIG. 1, the arrays can be preparedby loading oligonucleotides (0.1-50 ng/III) of different sequences orconcentrations into the wells of a 96 well microtiter source plate 20;the first well can be reserved for holding a matrix solution. Asubstrate 34, such as a pitted silicon chip substrate, can be placed onthe stage 26 of the robotics assembly 16 and can be aligned manually toorient the matrix of wells about a set of reference axes. The controlprogram executing on the data processor 12 can receive the coordinatesof the first well of the source plate 20. The robotic arm 24 can dip thepin assembly 38 into source plate 20 such that each of the 16 pins isdipped into one of the wells. Each vesicle can fill by capillary actionso that the full volume of the holding chamber contains fluid.Optionally, the program executing on the data processor 12 can directthe pressure controller to fill the interior chamber 58 of the pinassembly 38 with a positive bias pressure that will counteract, in part,the force of the capillary action to limit or reduce the volume of fluidthat is drawn into the holding chamber.

Optionally, the pin assembly 38 can be dipped into the same 16 wells ofthe source plate 20 and spotted on a second target substrate. This cyclecan be repeated on as many target substrates as desired. Next therobotic arm 24 can dip the pin assembly 38 in a washing solution, andthen dip the pin assembly into 16 different wells of the source plate20, and spot onto the substrate target offset a distance from theinitial set of 16 spots. Again this can be repeated for as many targetsubstrates as desired. The entire cycle can be repeated to make a 2×2array from each vesicle to produce an 8×8 array of spots (2×2elements/vesicle×16 vesicles=64 total elements spotted). However, itwill be apparent to anyone of ordinary skill in the art that processsuitable for forming arrays can be practiced with the present inventionwithout departing from the scope thereof.

Oligonucleotides of different sequences or concentrations can be loadedinto the wells of up to three different 384-well microtiter sourceplates; one set of 16 wells can be reserved for matrix solution. Thewells of two plates are filled with washing solution. Five microtiterplates can be loaded onto the stage of the robotic assembly 16. Aplurality of target substrates can be placed abutting an optional set ofbanking or registration pins disposed on the stage 26 and provided foraligning the target substrates along a set of reference axes. If thematrix and oligonucleotide are not pre-mixed, the pin assembly can beemployed to first spot matrix solution on all desired target substrates.In a subsequent step the oligonucleotide solution can be spotted in thesame pattern as the matrix material to re-dissolve the matrix.Alternatively, a sample array can be made by placing the oligonucleotidesolution on the wafer first, followed by the matrix solution, or bypre-mixing the matrix and oligonucleotide solutions.

After depositing the sample arrays onto the surface of the substrate,the arrays can be analyzed using any of a variety of means (e.g.,spectrometric techniques, such as UV/VIS, IR, fluorescence,chemiluminescence, NMR spectrometry or mass spectrometry). For example,subsequent to either dispensing process, sample loaded substrates can beplaced onto a MALDI-TOF source plate and held there with a set ofbeveled screw mounted polycarbonate supports. In one practice, the platecan be transferred on the end of a probe to be held onto a 1 μmresolution, 1″ travel xy stage (Newport) in the source region of atime-of-flight mass spectrometer. It will be apparent to one of ordinaryskill in the art that any suitable mass spectrometry tool can beemployed with the present invention without departing from the scopethereof.

Preferred mass spectrometer formats for use with the arrays describedherein include ionization (I) techniques including but not limited tomatrix assisted laser desorption (MALDI), continuous or pulsedelectrospray (ESI) and related methods (e.g. lonspray or Thermospray),or massive cluster impact (MCI); those ion sources can be matched withdetection formats including linear or non-linear reflectrontime-of-flight (TOF), single or multiple quadruple, single or multiplemagnetic sector, Fourier Transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof (e.g., ion-trap/time-of-flight). Forionization, numerous matrix/wavelength combinations (MALDI) or solventcombinations (ESI) can be employed. Subattomole levels of protein havebeen detected for example, using ESI (Valaskovic, G. A. et al., (1996)Science 273: 1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc118: 1662-1663) mass spectrometry.

Thus, it will be understood that in processes described herein acompletely non-contact, high-pressure spray or partial-contact, lowpressure droplet formation mode can be employed. In the latter, the onlycontact that will occur is between the droplet and the walls of the wellor a hydrophilic flat surface of the substrate 34. In neither practiceneed there be any contact between the needle tip and the surface.

Preferred Embodiments

In one preferred embodiment, a nucleic acid molecule can be covalentlyimmobilized on a silica support by functionalization of the support withan amino functionality (e.g., by derivatization of the support with areagent such as 3-aminopropyl-triethoxysilane (Aldrich Chemical Co.,Milwaukee, Wis.); see FIG. 7). Other functionalized oxysilanes ororthosilicates can be used, and are commercially available (e.g., fromGelest, Inc., Tullytown, Pa.). For example,3-mercaptopropyltriethoxy-silane can be used to functionalize a siliconsurface with thiol groups. The amino-functionalized silica can then bereacted with a heterobifunctional reagent such as N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB) (Pierce, Rockford, Ill.). Otherhomo- and hetero-bifunctional reagents which can be employed areavailable commercially, e.g., from Pierce. Finally, a nucleic acidfunctionalized with a thiol,group (e.g., at the 5′-terminus) iscovalently bound to the derivatized silica support by reaction of thethiol functionality of the nucleic acid molecule with the iodoacetylfunctionality of the support.

In certain embodiments, the nucleic acid can be reacted with thecross-linking reagent to form a cross-linker/nucleic acid conjugate,which is then reacted with a functionalized support to provide animmobilized nucleic acid. Alternatively, the cross-linker can becombined with the nucleic acid and a functionalized solid support in onepot to provide substantially simultaneous reaction of the cross-linkingreagent with the nucleic acid and the solid support. In this embodiment,it will generally be necessary to use a heterobifunctional cross-linker,i.e., a cross-linker with two different reactive functionalities capableof selective reaction with each of the nucleic acid and thefunctionalized solid support.

The methods provided herein are useful for producingspatially-addressable arrays of nucleic acids immobilized on insolublesupports. For example, the methods can be used to provide arrays ofdifferent nucleic acids immobilized on pins arranged in an array. Inanother embodiment, a photo-cleavable protecting group on the insolublesupport can be selectively cleaved (e.g., by photolithography) toprovide portions of a surface activated for immobilization of a nucleicacid. For example, a silicon surface, modified by treatment with3-mercaptopropyl-triethoxysilane to provide thiol groups, can be blockedwith a photocleavable protecting group (for examples of photocleavableprotecting groups, see, e.g., PCT Publication WO 92/10092, or McCray etal., (1989) Ann. Rev. Biophvs. Biorhys. Chem. 18:239-270), and beselectively deblocked by irradiation of selected areas of the surface,e.g., by use of a photolithography mask. A nucleic acid modified tocontain a thiol-reactive group can then be attached directly to thesupport, or, alternatively, a thiol-reactive cross-linking reagent canbe reacted with the thiol-modified support, followed by (orsubstantially simultaneously with) reaction with a nucleic acid toprovide immobilized nucleic acids. A nucleic acid base or sequence, onceimmobilized on a support according to the methods described herein, canbe further modified according to known methods. for example, the nucleicacid sequence can be lengthened by performing solid-phase nucleic acidsynthesis according to conventional techniques, including combinatorialtechniques.

Insoluble supports comprising nucleic acids are provided herein.Preferably the nucleic acids are covalently bound to a surface of theinsoluble support through at least one sulfur atom, i.e., the nucleicacids are covalently bound to the surface through a linker moiety whichincludes at least one sulfur atom. Such covalently bound nucleic acidsare readily produced by the methods described herein. The insolublesupports can be used in a variety of applications including those thatinvolve hybridization and sequencing. Exemplary applications areillustrated in the Examples.

In preferred embodiments, the covalently bound nucleic acids are presenton the surface of the insoluble support at a density of at least about20 fmol/mm², more preferably at least about 75 fmol/mm², still morepreferably at least about fmol/mm², yet more preferably at least about100 fmol/mm², and most preferably at least about 150 fmol/mm².

In another aspect, combinatorial libraries of immobilized nucleic acids,covalently bound to a solid support as described above are provided.

In still another aspect, a kit for immobilized nucleic acids on a solidsupport is provided. In one embodiment, the kit comprises an appropriateamount of: i) a thiol-reactive cross-linking reagent; and ii) asurface-modifying reagent for modifying a surface with a functionality(preferably other than a thiol) which can react with the thiol-reactivecross-linking reagent. The kit can optionally include an insolublesupport, e.g., a solid surface, e.g., magnetic microbeads, for use inimmobilized nucleic acids. The kit can also include a reagent formodifying a nucleic acid with a thiol functionality.

In another embodiment, the kit comprises a reagent for modifying thesurface of a support with a thiol moiety, and a thiol-reactivecross-linking reagent which can react with a thiol moiety of a support.In certain embodiments, the kit also includes an insoluble support,e.g., a solid surface, e.g., magnetic microbeads, for use inimmobilizing nucleic acids.

The kits described herein can also optionally include appropriatebuffers; containers for holding the reagents; and/or instructions foruse.

In yet another embodiment, the insoluble supports covalently bound withnucleic acids, e.g., the entire surface or spatially addressable orpre-addressable array formats, can be used in a variety of solid phasenucleic acid chemistry applications, including but not limited tonucleic acid synthesis (chemically and enzymatically), hybridizationand/or extension, and in diagnostic methods based in nucleic aciddetection and polymorphism analyses (see, e.g., U.S. Pat. No.5,605,798). Accordingly, further provided herein are methods of reactingnucleic acid molecules in which the nucleic acid molecules areimmobilized on a surface either by reacting a thiol-containingderivative of the nucleic acid molecule with an insoluble supportcontaining a thiol-reactive group or by reacting a thiol-containinginsoluble support with a thiol-reactive group-containing derivative ofthe nucleic acid molecule and thereafter further reacting theimmobilized nucleic acid molecules.

In a particular embodiment, the immobilized nucleic acid is furtherreacted by hybridizing with a nucleic acid that is complementary to theimmobilized nucleic acid or a portion thereof. In another embodiment,the immobilized nucleic acid is further reacted by extension of anucleic acid that is hybridized to the immobilized nucleic acid or aportion thereof. Extension reactions such as these can be used, forexample, in methods of sequencing DNA molecules that are immobilized toan insoluble support using the processes described herein. Thus, alsoprovided herein are methods of determining the sequence of a DNAmolecule on a substrate in which a thiol-containing derivative of theDNA molecule is immobilized on the surface of an insoluble supportcontaining thiol-reactive groups and hybridized with a single-strandednucleic acid complementary to a portion of the immobilized DNA prior tocarrying out DNA synthesis in the presence of one or moredideoxynucleotides.

The present invention is further illustrated by the following Examples,which are intended merely to further illustrate and should not beconstrued as limiting. The entire contents of all the of the references(including literature references, issued patents, published patentapplications, and co-pending patent applications) cited throughout thisapplication are hereby expressly incorporated by reference.

EXAMPLE 1 High Density Attachment of DNA to Silicon Wafers

Materials and Methods

All reagents, unless otherwise noted, were obtained from AldrichChemical, Milwaukee, Wis.

Silicon Surface Preparation

Silicon wafers were washed with ethanol, flamed over bunsen burner, andimmersed in an anhydrous solution of 25% (by volume)3-aminopropyltriethoxysilane in toluene for 3 hours. The silane solutionwas then removed, and the wafers were washed three times with tolueneand three times with dimethyl sulfoxide (DMSO). The wafers were thenincubated in a 10 mM anhydrous solution of N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) (Pierce Chemical, Rockford, Ill.) in anhydrousDMSO. Following the reaction, the SIAB solution was removed, and thewafers were washed three times with DMSO.

Since it was impossible to monitor the condensation of SIAB and theamino group while on the solid support of the wafer, the reaction wasperformed in solution to determine the optimal reaction time. Thin layerchromatography (TLC) (glass backed silica plates with a 254 nmfluorescent indicator) (Baker, Phillipsburg, NF) was employed using 95:5chloroform:methanol (Baker, Phillipsburg, NJ) which enabled separationof the two starting materials. It was possible to visualize the SIABstarting material under long wave ultraviolet light (302 nm);3-aminopropyltriethoxysilane was not active under ultraviolet light,therefore, the plate was sprayed with a solution of ninhydrin whichreacts with primary amines to reveal a purple spot upon heating. Amicroscale reaction was run in chloroform/DMSO using a slight molarexcess of SIAB in comparison to 3-aminopropyltriethoxysilane andmonitored with the above mentioned TLC conditions.

Oligonucleotide Modifications

Reduction of the disulfide from 3′- or 5′-disulfide-containingoligodeoxynucleotides (Operon Technologies, Alameda, Calif. or OligoEtc., Wilsonville, Oreg.) was monitored using reverse-phase FPLC(Pharmacia, Piscataway, N.J.); a shift can be seen in the retention timeof the oligodeoxynucleotide upon cleavage of the disulfide. Variousreduction methods were investigated to determine the optimal conditions.In one case, the disulfide-containing oligodeoxynucleotide (31.5 nmol,0.5 mM) was incubated with dithiothreitol (DTT) (Pierce Chemical,Rockford, Ill.) (6.2 mmol, 100 mM) as pH 8.0 and 37° C. With thecleavage reaction essentially complete, the free thiol-containingoligodeoxynucleotide was isolated using a Chromaspin-10 column(Clontech, Palo Alto, Calif.) since DTT may compete in the subsequentreaction. Alternatively, tris-(2-carboxyethyl) phosphine (TCEP) (PierceChemical, Rockford, Ill.) has been used to cleave the disulfide. Thedisulfide-containing oligodeoxynucleotide (7.2 nmol, 0.36 mM) wasincubated with TCEP in pH 4.5 buffer at 37° C. It is not necessary toisolate the product following the reaction since TCEP does notcompetitively react with the iodoacetamido functionality. Varyingconcentrations of TCEP were used for the cleavage reaction to determinethe optimal conditions for the conjugation reaction.

Probe Coupling

To each wafer which had been derivatized to contain the iodoacetamidofunctionality as described above was added a 10 mM aqueous solution ofthe free-thiol containing oligodeoxynucleotide in 100 mM phosphatebuffer, pH 8; the reaction was allowed to proceed for a minimum of fivehours at room temperature in 100% relative humidity. Following thereaction, the oligodeoxynucleotide solution was removed, and the waferswere washed two times in 5×SSC buffer (75 mM sodium citrate, 750 mMsodium chloride, pH 7) with 50% formamide (USB, Cleveland, Ohio) at 65°C. for 1 hour each.

Radiochemical Determination of Probe Density

In order to determine the amount of DNA covalently attached to a surfaceor the amount of a complementary sequence hybridized, radiolabeledprobes were employed. In cases where a 5′-disulfide-containingoligodeoxynucleotide was to be immobilized, the 3′-terminus wasradiolabeled using terminal transferase enzyme and a radiolabeleddideoxynucleoside triphosphate; in a standard reaction, 15 pmol (0.6 μM)of the 5′-disulfide-containing oligodeoxynucleotide was incubated with50 μCi (16.5 pmol, 0.66 μM) of [α-³²P] dideoxyadenosine-5′triphosphate(ddATP) (Amersham, Arlington Height, Ill.) in the presence of 0.2 mM2-mercaptoethanol. Upon the addition of 40 units of the terminaldeoxynucleotidyl transferase enzyme (USB, Clevetand, Ohio), the reactionwas allowed to proceed for one hour at 37° C. After this time, thereaction was stopped by immersion of the vial in 75° C. water bath forten minutes, and the product was isolated using a Chromaspin-10 column(Clontech, Palo Alto, Calif.). Similarly, a 5′-disulfide-containingoligodeoxynucleotide was radiolabeled with ³⁵S.

In cases where a 3′-disulfide-containing oligodeoxynucleotide was to beimmobilized, the 5′-terminus was radiolabeled using T4 polynucleotidekinase and a radiolabeled nucleoside triphosphate. For example, 15 pmol(0.6 μM) of the 3′-disulfide-containing oligodeoxynucleotide wasincubated with 50 μCi (16.5 pmol, 0.66 μM) of [λ³²P]adenosine-5′triphosphate (ATP) (Amersham, Arlington Height, Ill.) in thepresence of 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂ 10 mM 2-mercaptoethanol.Following the addition of 40 units of T4 polynucleotide kinase, thereaction was allowed to proceed for 1 hour at 37° C. The reaction wasstopped by immersion of the vial in a 75° C. water bath for ten minutes;the product was then isolated using a Chromaspin-10 column (Clontech,Palo Alto, Calif.).

To determine the density of covalently immobilized probe, thedisulfide-containing oligodeoxynucleotide of choice was added to a traceamount of the same species than had been radiolabeled as describedabove. The disulfide was cleaved, the probe was immobilized oniodoacetamido-functionalized wafers, the wafers were washed, and thenexposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale,Calif.). For each different oligodeoxynucleotide utilized, referencespots were made on polystyrene in which the molar amount ofoligodeoxynucleotide was known; these reference spots were exposed tothe phosphorimager screen as well. Upon scanning the screen, thequantity (in moles) of oligodeoxynucleotide bound to each chip wasdetermined by comparing the counts to the specific activities of thereferences.

Hybridization and Efficiency

To a wafer that had been functionalized with an immobilized probe wasadded a solution of a complementary sequence (10 μM) in 1M NaCl and TEbuffer. The wafer and solution were heated to 75° C. and allowed to coolto room temperature over 3 hours. After this time, the solution wasremoved, and the wafer was washed two times with TE buffer.

To determine the amount of oligonucleotide hybridized, immobilization ofthe probe was first carried out as described above except that the probewas labeled with ³⁵S rather than ³²P. The density of immobilized probewas determined with the phosphorimager. Next, the same wafer wasincubated in TE buffer, 1M NaCl, and its complementary strand (10 μM)which had been radiolabeled with ³²P. Hybridization was carried out aspreviously described. Following a wash to remove non-specific binding,the wafer and reference were exposed to a phosphorimager screen with apiece of copper foil between the screen and the wafer. The copper foilserves to block the signal from ³⁵S, while allowing the ³²P signal topass freely. The molar amount of hybridized oligonucleotide is thendetermined, thus revealing the percent of covalently immobilized probethat is available for hybridization.

MALDI-TOF Mass Spectrometric Analysis

As described above, wafers containing non-radiolabeled immobilizedoligodeoxynucleotide (name: TCUC; sequence: GAATTCGAGCTCGGTACCCGG;molecular weight; 6455Da; SEQ ID NO. 1) were synthesized, and acomplementary sequence (name: MJM6; sequence: CCGGGTACCGAGCTCGAATTC;molecular weight: 6415Da; SEQ ID NO. 2) was hybridized. The wafers werewashed in 50 mM ammonium citrate buffer for cation exchange to removesodium and potassium ions on the DNA backbone (Pieles, U. et al., (1993)Nucl. Acids Res., 21:3191-3196). A matrix solution of 3-hydroxypicolinicacid (3-HPA, 0.7 M in 50% acetonitrile, 10% ammonium citrate; Wu, K. J.,et al. (1993) Rapid Commun. Mass Spectrom., 7:142-146) was spotted ontothe wafer and allowed to dry at ambient temperature. The wafers wereattached directly to the sample probe of a Finnigan MAT (Bremen,Germany) Vision 2000 reflectron TOF mass spectrometer using a conductingtape. The reflectron possesses a 5 keV ion source and 20 keVpost-acceleration; a nitrogen laser was employed; and all spectra weretaken in the positive ion mode.

Results

Surface Chemistry

Employing standard silicon dioxide modification chemistry, a siliconwafer was reacted with 3-aminopropyltriethoxysilane to produce a uniformlayer of primary amino groups on the surface. As shown in FIG. 7, thesurface was then exposed to a heterobifunctional crosslinker resultingin iodoacetamido groups on the surface. It was possible to determine theoptimal reaction time of this reaction in solution using TLC. The SIABcrosslinker was visualized under long wave ultraviolet light (302 nm) toreveal a spot with an R_(f) value of 0.58. 3-aminopropyltriethoxysilanewas not active under ultraviolet light, therefore, ninhydrin was used toreveal a purple spot indicating the presence of a primary amine at thebaseline. A microscale reaction was run using a slight molar excess ofSIAB in comparison to 3-aminopropyltriethoxysilane; TLC analysis afterapproximately one minute revealed a new spot visible under long waveultraviolet light with an R_(f) value of 0.28. There was no evidence ofa purple spot upon spraying with ninhydrin, thus all the3-aminopropyltriethoxysilane starting material had been consumed in thereaction. UV light also revealed the excess SIAB which remainedfollowing the reaction. From these results, it was determined thereaction is complete after approximately one minute. In all cases, theiodoacetamido-functionalized wafers were used immediately to minimizehydrolysis of the labile iodoacetamido-functionality. Additionally, allfurther wafer manipulations were performed in the dark since theiodoacetamido-functionality is light sensitive.

Disulfide reduction of the modified oligonucleotide was monitored byobserving a shift in retention time on reverse-phase FPLC. It wasdetermined that after five hours in the presence of DTT (100 mM) or TCEP(10 mM), the disulfide was fully reduced to a free thiol. If the DTTreaction was allowed to proceed for a longer time, an oligonucleotidedimer formed in which pairs of free thiols had reacted. Suchdimerization was also observed when the DTT was removed following thecompletion of the cleavage reaction. This dimerization was not observedwhen TCEP was employed as the cleavage reagent since this reaction isperformed at pH 4.5, thus the free thiols were fully protonatedinhibiting dimerization.

Immediately following disulfide cleavage, the modified oligonucleotidewas incubated with the iodacetamido-functionalized wafers. To ensurecomplete thiol deprotonation, the coupling reaction was performed at pH8.0. The probe surface density achieved by this chemistry of siliconwafers was analyzed using radiolabeled probes and a phosphorimager. Theprobe surface density was also monitored as a function of the TCEPconcentration used in the disulfide cleavage reaction (FIG. 8). Using 10mM TCEP to cleave the disulfide and the other reaction conditionsdescribed above, it was possible to reproducibly yield a surface densityof 250 fmol per square mm of surface. Identical experiments as describedabove were performed except that the oligonucleotide probe lacked athiol modification; surface densities of less than 5 fmol per square mmof surface proved that non-specific binding is minimal and that probecoupling most likely occurred as proposed in FIG. 7.

Hybridization

After attaching ³⁵S-labeled probes to the surface of wafers anddetermining conjugation density as described above, hybridization of³²P-labeled oligonucleotides was carried out; hybridization efficiencyand density were determined using the phosphorimager and copper foil. Itwas determined experimentally that copper foil blocks 98.4% of an ³Ssignal, while fully allowing a ³²P signal to be detected. Thecomplementary sequence reproducibly hybridized to yield 105 fmol persquare mm of surface; this corresponds to approximately 40% of theconjugated probes available for hybridization. Similarly, anon-complementary sequence was employed in this scheme yielding lessthan 5 fmol per square mm of surface in non-specific binding.

It is hypothesized that stearic interference between the tightly packedoligonucleotide on the flat surface inhibits hybridization efficiencieshigher that 40%. With this in mind, a spacer molecule was incorporatedbetween the terminus of the hybridizing region of the oligonucleotideand the support. The chosen spacers were a series of poly dT sequencesranging in length from 3 to 25. Upon examination of these samples withradiolabels and the phosphorimager, it was determined that 40% was stillthe maximum hybridization that could be achieved.

MALDI-TOF MS Analysis

Wafers were functionalized with probes, complementary sequences werehybridized, and the samples were analyzed under standard MALDIconditions as described above. Analysis revealed that only the annealedstrand (MJM6) was observed in the mass spectrum with an experimentalmass-to-charge ratio of 6415.4; the theoretical mass-to-charge ratio is6415 (FIG. 9). Since there was no signal at a mass-to-charge ratio of6455, it was determined that the wafer-conjugated strand (TCUC) was notdesorbed thus the iodoacetamido linkage was stable enough to withstandthe laser and remain intact. There was an additional signal observed ata mass-to-charge ration of 6262.0. This signal results from adepurination of guanosines since it is known that DNA is susceptible tothe loss of purine bases during the MALDI process, (Nordoff, E., et al.,(1992) Rapid Commun. Mass Spectrom. 6:771-776). The sample crystals onthe wafer were not homogeneously distributed, thus it was necessary tohunt for a good spot . Because of this non-homogeneity, the massresolution varied, but it generally ranged from 200-300 for the desorbedoligonucleotide in the mass spectra. In one set of experiments,non-complementary sequences were hybridized to the wafer; following awash as previously described, analysis by MALDI-TOF MS revealed thatminimal non-specific annealing had taken place since no signal wasdetected.

EXAMPLE 2 Immobilization of Amplified DNA Targets to Silicon Wafers

The SIAB-conjugated silicon wafers were also used to analyze specificfree thiol-containing DNA fragments of a particular amplified DNA targetsequence.

As shown in FIG. 10, a 23-mer oligodeoxynucleotide containing a5′-disulfide linkage [purchased from Operon Technologies; SEQ ID NO: 3]that is complementary to the 3′-region of a 112 bp human genomic DNAtemplate [Genebank Acc. No.: Z52259; SEQ ID NO: 4] was used as a primerin conjunction with a commercially available 49-mer primer, which iscomplementary to a portion of the 5′-end of the genomic DNA [purchasedfrom Operon Technologies; SEQ ID NO: 5], in PCR reactions to amplify a135 bp DNA product containing a 5′-disulfide linkage attached to onlyone strand of the DNA duplex [SEQ ID NO: 6].

The PCR amplification reactions were performed using the AmplitaqGoldKit [Perkin Elmer Cataolog No. N808-0249]. Briefly, 200 ng 112 bphuman genomic DNA template was incubated with 10 μM of 23-mer primer and8 μM of commercially available 49-mer primer, 10 mM dNTPs, 1 unit ofAmplitaq Gold DNA polymerase in the buffer provided by the manufacturerand PCR was performed in a thermocycler.

The 5′-disulfide bond of the resulting PCR product was fully reducedusing 10 mM TCEP as described in EXAMPLE 1 to generate a free 5′-thiolgroup. The DNA strand containing free-thiol group was conjugated to thesurface of the silicon wafer through the SIAB linker essentially asoutlined in FIG. 7.

The silicon wafer conjugated with the 135 bp thiol-containing DNA wasincubated with a complementary 12-mer oligonucleotide [SEQ ID NO: 7] andspecifically hybridized DNA fragments were detected using MALDI-TOF MSanalysis. The mass spectrum revealed a signal with an observedexperimental mass-to-charge ratio of 3618.33; the theoreticalmass-to-charge ratio of the 12-mer oligomer sequence is 3622.4 Da.

Thus, a specific DNA target molecule that contain a 5′-disulfide linkagecan be amplified. The molecules are immobilized on a SIAB-derivatizedsilicon wafer using the methods described herein and specificcomplementary oligonucleotides may be hybridized to these targetmolecules and detected using MALDI-TOF MS analysis.

EXAMPLE 3 Spectrochip Mutant Detection in ApoE Gene

This example describes the hybridization of an immobilized template,primer extension and mass spectrometry for detection of the wildtype andmutant Apolipoprotein E gene for diagnostic purposes. This exampledemonstrates that immobilized DNA molecules containing a specificsequence can be detected and distinguished using primer extension ofunlabeled allele specific primers and analysis of the extension productsusing mass spectrometry.

A 50 base synthetic DNA template complementary to the coding sequence ofallele 3 of the wildtype apolipoprotein E gene:

5′-GCCTGGTACACTGCCAGGCGCTTCTGCAGGTCATCGGCATCGCGGAGGAG-3′ [SEQ ID NO: 17]

or complement to the mutant apolipoprotein E gene carrying a G→Atransition at codon 158:

5′-GCCTGGTACACTGCCAGGCACTTCTGCAGGTCATCGGCATCGCGGAGGAG-3′ [SEQ ID NO: 18]

containing a 3′-free thiol group was coupled to separateSIAB-derivatized silicon wafers essentially as outlined in FIG. 7 and asdescribed in Examples 1 and 2.

A 21-mer oligonucleotide primer:

5′-GATGCCGATGACCTGCAGAAG-3′ [SEQ ID NO: 19] was hybridized to each ofthe immobilized templates and the primer was extended using acommercially available kit [e.g., Sequenase or Thermosequenase, U.S.Biochemical Corp]. The addition of Sequenase DNA polymerase orThermosequenase DNA polymerase in the presence of threedeoxyribonucleoside triphosphates (dNTPs; dATP, dGTP, dTTP) anddideoxyribonucleoside cytosine triphosphate (ddCTP) in buffer accordingto the instructions provided by the manufacturer resulted in a singlebase extension of the 21-mer primer bound to the immobilized templateencoding the wildtype apolipoprotein E gene and a three base extensionof the 21-mer primer bound to the immobilized template encoding themutant form of apolipoprotein E gene.

The wafers were analyzed by mass spectrometry as described herein. Thewildtype apolipoprotein E sequence results in a mass spectrum thatdistinguishes the primer with a single base extension (22-mer) with amass-to-charge ratio of 6771.17 Da (the theoretical mass to charge ratiois 6753.5 Da) from the original 21-mer primer with a mass-to-chargeratio of 6499.64 Da. The mutant apolipoprotein E sequence results in amass spectrum that distinguishes the primer with a three base extension(24-mer) with a mass-to-charge ratio of 7386.9 (the theoretical masscharge is 7386.9) from the original 21-mer primer with a mass to chargeration of 6499.64 Da.

EXAMPLE 4 Preparation of DNA Arrays Using Serial and Parallel DispensingTools

Robot-driven serial and parallel pL-nL dispensing tools were used togenerate 10-10³ element DNA arrays on <1″ square chips with flat orgeometrically altered (e.g. with wells) surfaces for matrix assistedlaser desorption ionization mass spectrometry analysis. In the former, a‘piezoelectric pipette’ (70 μm id capillary) dispenses single ormultiple ˜0.2 nL droplets of matrix, and then analyte, onto the chip;spectra from as low as 0.2 fmol of a 36-mer DNA have been acquired usingthis procedure. Despite the fast (<5 sec) evaporation, micro-crystals of3-hydroxypicolinic acid matrix containing the analyte are routinelyproduced resulting in higher reproducibility than routinely obtainedwith larger volume preparations; all of 100 five fmol spots of a 23-merin 800 μm wells yielded easily interpreted mass spectra, with 99/100parent ion signals having signal to noise ratio of >5. In a secondapproach, probes from 384 well microtiter plate are dispensed 16 at atime into chip wells or onto flat surfaces using an array of springloaded pins which transfer ˜20 nL to the chip by surface contact; MSanalysis of array elements deposited with the parallel method arecomparable in terms of sensitivity and resolution to those made with theserial method.

Description of the Piezoelectric Serial Dispenser

The experimental system developed from a system purchased from MicrodropGmbH, Norderstedt Germany and can include a piezoelectric element driverwhich sends a pulsed signal to a piezoelectric element bonded to andsurrounding a glass capillary which holds the solution to be dispensed;a pressure transducer to load (by negative pressure) or empty (bypositive pressure) the capillary; a robotic xyz stage and robot driverto maneuver the capillary for loading, unloading, dispensing, andcleaning, a stroboscope and driver pulsed at the frequency of the piezoelement to enable viewing of ‘suspended’ droplet characteristics;separate stages for source and designation plates or sample targets(i.e. Si chip); a camera mounted to the robotic arm to view loading todesignation plate; and a data station which controls the pressure unit,xyz robot, and piezoelectric driver.

Description of the Parallel Dispenser

The robotic pintool consists of 16 probes housed in a probe block andmounted on an X Y, Z robotic stage. The robotic stage was a gantrysystem which enables the placement of sample trays below the arms of therobot. The gantry unit itself is composed of X and Y arms which move 250and 400 mm, respectively, guided by brushless linear servo motors withpositional feedback provided by linear optical encoders. A lead screwdriven Z axis (50 mm vertical travel) is mounted to the xy axis slide ofthe gantry unit and is controlled by an in-line rotary servo motor withpositional feedback by a motor-mounted rotary optical encoder. The workarea of the system is equipped with a slide-out tooling plate that holdsfive microtiter plates (most often, 2 plates of wash solution and 3plates of sample for a maximum of 1152 different oligonucleotidesolutions) and up to ten 20×20 mm wafers. The wafers are placedprecisely in the plate against two banking pins and held secure byvacuum. The entire system is enclosed in plexi-glass housing for safetyand mounted onto a steel support frame for thermal and vibrationaldamping. Motion control is accomplished by employing a commercial motioncontroller which was a 3-axis servo controller and is integrated to acomputer; programming code for specific applications is written asneeded.

Samples were dispensed with the serial system onto several surfaceswhich served as targets in the MALDI TOF analysis including [1] A flatstainless steel sample target as supplied for routine use in a ThermoBioanalysis Vision 2000; [2] the same design stainless steel target withmicromachined nanopits; [3] flat silicon (Si) wafers; [4] polished flatSi wafers; [5] Si wafers with rough (3-6 pLm features) pits; [6](a)12×12 or ((b) 18×18) mm Si chips with (a) 10×10 (or (b) 16×16) arrays ofchemically etched wells, each 800×8001 lm on a side with depths rangingfrom 99-400 (or(b) 120) micrometer, pitch (a) 1.0 (or(b) 1.125) mm; [7]15×15 mm Si chips with 28×28 arrays of chemically etched wells, each450×450 micrometer on a side with depths ranging from 48-300 micrometer,pitch 0.5 mm; [8]flat polycarbonate or other plastics; 19] gold andother metals; [10] membranes; [11] plastic surfaces sputtered with goldor other conducting materials. The dispensed volume is controlled from10⁻¹⁰ to 10³¹ ⁶ L by adjusting the number of droplets dispensed.

Sample Preparation and Dispensing

1. Serial

Oligonucleotides (0.1-50 ng/microliter of different sequence orconcentrations were loaded into wells of a 96 well microtiter plate; thefirst well was reserved for matrix solution. A pitted chip (target 6a inMALDI targets' section) was placed on the stage and aligned manually.Into the (Windows-based) robot control software were entered thecoordinates of the first well, the array size (ie number of spots in xand y) and spacing between elements, and the number of 0.2 nL drops perarray element. The capillary was filled with ˜10 microL rinse H₂O,automatically moved in view of a strobe light-illuminated camera forchecking tip integrity and cleanliness while in continuous pulse mode,and emptied. The capillary was then filled with matrix solution, againchecked at the stroboscope, and then used to spot an array onto flat orpitted surfaces. For Reproductability studies in different MS modes,typically a 10×10 array of 0.2-20 nL droplets were dispensed. Thecapillary was emptied by application of positive pressure, optionallyrinsed with H₂O, and led to the source oligo plate where ˜5 μL of0.05-2.0 μM synthetic oligo were drawn. The capillary was then rasteredin series over each of the matrix spots with 0.2-20 nL aqueous solutionadded to each.

2. Parallel

Parallel Programs were written to control array making by offsetprinting; to make an array of 64 elements on 10 wafers, for example, thetool was dipped into 16 wells of a 384 well DNA source plate, moved tothe target (e.g. Si, plastic, metal), and the sample spotted by surfacecontact. The tool was then dipped into the same 16 wells and spotted onthe second target; this cycle was repeated on all ten wafers. Next thetool was dipped in washing solution, then dipped into 16 different wellsof the source plate, and spotted onto the target 2.25 mm offset from theinitial set of 16 spots; again this was repeated on all 10 wafers; theentire cycle was repeated to make a 2×2 array from each pin to producean 8×8 array of spots (2×2 elements/pin×16 pins=64 total elementsspotted).

To make arrays for MS analysis, olegonucleotides of different sequencesor concentrations were loaded into the wells of up to three different384-well microtiter plates, one set of 16 wells was reserved for matrixsolution. The wells of two plates were filled with washing solution. Thefive microtiter plates were loaded onto the slide-out tooling plate. Tenwafers were placed abutting the banking pins on the tooling plate, andthe vacuum turned on. In cases where matrix and oligonucleotide were notpre-mixed, the pintool was used to spot matrix solution first on alldesired array elements of the ten wafers. For this example, a 16×16array was created, thus the tool must spot each of the ten wafers 16times, with an offset of 1.125 mm. Next, the oligonucleotide solutionwas spotted in the same pattern to re-dissolve the matrix. Similarly, anarray could be made by placing the oligonucleotide solution on the waferfirst, followed by the matrix solution, or by pre-mixing the matrix andoligonucleotide solutions.

Mass Spectrometry

Subsequent to either dispensing scheme, loaded chips were held onto aMALDI-TOF source plate with a set of beveled screw mountedpolycarbonated supports. The plate was transferred on the end of a probeto be held onto a 1 μm resolution, 1″ travel xy stage (Newport) in thesource region of a time-of-flight mass spectrometer. The instrument,normally operated with 18-26 kV extraction, could be operated in linearor curved field reflectron mode, and in continuous or delayed extractionmode.

RESULTS

Serial Dispensing with the Piezoelectric Pipette

While delivery of a saturated 3HPA solution can result in tip cloggingas the solvent at the capillary-air interface evaporates, pre-mixing DNAand matrix sufficiently dilutes the matrix such that it remains insolution while stable sprays which could be maintained until thecapillary was emptied were obtained; with 1:1 diluted (in H₂O) matrixsolution, continuous spraying for >>10 minutes was possible. Turning offthe piezo element so that the capillary sat inactive for >5 minutes, andreactivating the piezo element also did not result in a cloggedcapillary.

Initial experiments using stainless steel sample targets as provided byFinnigan Vision 2000 MALDI-TOF system run in reflectron mode utilized apre-mixed solution of the matrix and DNA prior to dispensing onto thesample target. In a single microtiter well, 50 μL saturated matrixsolution, 25 μL of a 51 μL solution of the 12-mer (ATCG)3, and 25 μL ofa 51 μL solution of the 28-mer (ATCG)7 were mixed. A set of 10×10 arraysof 0.6 μL drops was dispensed directly onto a Finnigan Vision 2000sample target disk; MALDI-TOF mass spectrum was obtained from a singlearray element which contained 750 attomoles of each of the twooligonucleotides. Interpretable mass spectra has been obtained for DNAsas large as a 53-mer (350 amol loaded, not shown) using this method.

Mass spectra were also obtained from DNAs microdispensed into the wellsof a silicon chip. FIG. 11 shows a 12×12 mm silicon chip with 100chemically etched wells; mask dimensions and etch time were set suchthat fustum (i.e., inverted flat top pyramidal) geometry wells with800×800 μm (top surface) and 100 μm depth were obtained. Optionally, thewells can be roughed or pitted. As described above, the hip edge wasaligned against a raised surface on the stage to define the x and ycoordinate systems with respect to the capillary. (Alternatives includeoptical alignment, artificial intelligence pattern recognition routines,and dowel-pin based manual alignment). Into each well was dispensed 20droplets (˜5 nL) of 3-HPA matrix solution without analyte; for the 50%CH₃CN solution employed, evaporation times for each droplet were on theorder of 5-10 seconds. Upon solvent evaporation, each microdispensedmatrix droplet as viewed under a 120× stereomicroscope generallyappeared as an amorphous and ‘milky’ flat disk; such appearances areconsistent with those of droplets from which the FIG. 3b spectrum wasobtained. Upon tip emptying, rinsing, and refilling with a 1.4 μmaqueous solution of a 23-mer DNA (M_(r)(calc)=6967 Da), the capillarywas directed above each of the 100 spots of matrix where 5 nL of theaqueous DNA solution was dispensed directly on top of the matrixdroplets. Employing visualization via a CCD camera, it appeared that theaqueous analyte solution mixed with and re-dissolved the matrix(complete evaporation took ˜10 sec at ambient temperature and humidity).The amorphous matrix surfaces were converted to true micro-crystallinesurfaces, with crystalline features on the order of <1 μm.

Consistent with the improved crystallization afforded by the matrixre-dissolving method, mass spectrum acquisition appeared morereproducible than with pre-mixed matrix plus analyte solutions; each ofthe 100 five fmol spots of the 23-mer yielded interpreted mass spectra(FIG. 12), with 99/100 parent ion signals having signal to noise ratiosof >5; such reproducibility was also obtained with the flat silicon andmetallic surfaces tried (not shown). The FIG. 12 spectra were obtainedon a linear TOF instrument operated at 26 kV. Upon internal calibrationof the top left spectrum (well ‘k1’) using the singly and doubly chargedmolecular ions, and application of this calibration file to all other 99spectra as an external calibration (FIG. 13), a standard deviation of <9Da from the average molecular weight was obtained, corresponding to arelative standard deviation of ˜0.1%.

Parallel Dispensing with the Robotic Pintool

Arrays were made with offset printing as described above. The velocityof the X and Y stages are 35 inches/sec, and the velocity of the Z stageis 5.5 inches/sec. It is possible to move the X and Y stages at maximumvelocity to decrease the cycle times, however the speed of the Z stageis to be decreased prior to surface contact with the wafer to avoiddamaging it. At such axes speeds, the approximate cycle time to spot 16elements (one tool impression of the same solutions) on all ten wafersis 20 seconds, so to make an array of 256 elements would take ˜5.3minutes. When placing different oligonucleotide solutions on the array,an additional washing step much be incorporated to clean the pin tipprior to dipping in another solution, thus the cycle time would increaseto 25 seconds or 6.7 minutes to make 10 wafers.

Sample delivery by the tool was examined using radio-labeled solutionsand the phosphorimager as described previously; it was determined thateach pin delivers approximately 1 nL of liquid. The spot-to-spotreproducibility is high. An array of 256 oligonucleotide elements ofvarying sequence and concentration was made on flat silicon wafers usingthe pintool, and the wafer was analyzed by MALDI-TOF MS.

EXAMPLE 5 Use of High Density Nucleic Acid Immobilization to GenerateNucleic Acid Arrays

Employing the high density attachment procedure described in EXAMPLE 1,an array of DNA oligomers amenable to MALDI-TOF mass spectrometryanalysis was created on a silicon wafer having a plurality of locations,e.g., depressions or patches, on its surface. To generate the array, afree thiol-containing oligonucleotide primer was immobilized only at theselected locations of the wafer [e.g., see FIG. 14]. Each location ofthe array contained one of three different oligomers. To demonstratethat the different immobilized oligomers could be separately detectedand distinguished, three distinct oligonucleotides of differing lengthsthat are complementary to one of the three oligomers were hybridized tothe array on the wafer and analyzed by MALDI-TOF mass spectrometry.

Oligodeoxynucleotides

Three sets of complementary oligodeoxynucleotide pairs were synthesizedin which one member of the complementary oligonucleotide pair contains a3′- or 5′-disulfide linkage [purchased from Operon Technologies orOligos, Etc.]. For example, Oligomer 1 [d(CTGATGCGTCGGATCATCTTTTTT-SS);SEQ ID NO: 8] contains a 3′-disulfide linkage whereas Oligomer 2[d(SS-CCTCTTGGGAACTGTGTAGTATT); a 5′-disulfide derivative of SEQ ID NO:3] and Oligomer 3 [d(SS-GAATTCGAGCTCGGTACCCGG); a 5′-disulfidederivative of SEQ ID NO: 1] each contain a 5′-disulfide linkage.

The oligonucleotides complementary to Oligomers 1-3 were designed to beof different lengths that are easily resolvable from one another duringMALDI-TOF MS analysis. For example, a 23-mer oligonucleotide [SEQ ID NO:9] was synthesized complementary to a portion of Oligomer 1, a 12-meroligonucleotide [SEQ ID NO: 7] was synthesized complementary to aportion of Oligomer 2 and a 21-mer [SEQ ID NO: 2; sequence denoted“MJM6” in EXAMPLE 1] was synthesized complementary to a portion ofOligomer 3. In addition, a fourth 29-mer oligonucleotide [SEQ ID NO: 10]was synthesized that lacks complementarity to any of the threeoligomers. This fourth oligonucleotide was used as a negative control.

Silicon Surface Chemistry and DNA Immobilization

(a) 4×4 (16-location) Array

A 2×2 cm² silicon wafer having 256 individual depressions or wells inthe form of a 16×16 well array was purchased from a commercial supplier[Accelerator Technology Corp., College Station, Tex.]. The wells were800×800 μm², 120 μm deep, on a 1.125 pitch. The silicon wafer wasreacted with 3-aminopropyltriethoxysilane to produce a uniform layer ofprimary amines on the surface and then exposed to the heterobifunctionalcrosslinker SIAB resulting in iodoacetamido functionalities on thesurface [e.g., see FIG. 7].

To prepare the oligomers for coupling to the various locations of thesilicon array, the disulfide bond of each oligomer was fully reducedusing 10 mM TCEP as depicted in EXAMPLE 1, and the DNA resuspended at afinal concentration of 10 μM in a solution of 100 mM phosphate buffer,pH 8.0. Immediately following disulfide bond reduction, the free-thiolgroup of the oligomer was coupled to the iodoacetamido functionality at16 locations on the wafer using the probe coupling conditionsessentially as described in FIG. 7. To accomplish the separate couplingat 16 distinct locations of the wafer, the entire surface of the waferwas not flushed with an oligonucleotide solution but, instead, an ˜30-nlaliquot of a predetermined modified oligomer was added in parallel toeach of 16 locations (i.e., depressions) of the 256 wells on the waferto create a 4×4 array of immobilized DNA using a pin tool as describedherein (see e.g., the Detailed Description and Example 4 providedherein).

Thus, as shown in FIG. 14, one of modified Oligomers 1-3 was covalentlyimmobilized to each of 16 separate wells of the 256 wells on the siliconwafer thereby creating a 4×4 array of immobilized DNA. For example,Oligomer 1 was conjugated at a well position in the upper left handcorner of the 4×4 array and Oligomer 2 was conjugated to the adjacentlocation, and so forth. An illustration of the completed array is shownin FIG. 14.

In carrying out the hybridization reaction, the three complementaryoligonucleotides and the negative control oligonucleotide were mixed ata final concentration of 10 μM for each oligonucleotide in 1 ml of TEbuffer [10 mM Tris-HCl, pH 8.0, 1 mM EDTA] supplemented with 1 M NaCl,and the solution was heated at 65° C. for 10 min. Immediatelythereafter, the entire surface of the silicon wafer was flushed with 800μl of the heated oligonucleotide solution. The complementaryoligonucleotides were annealed to the immobilized oligomers byincubating the silicon array at ambient temperature for 1 hr, followedby incubation at 4° C. for at least 10 min. Alternatively, theoligonucleotide solution can be added to the wafer which is then heatedand allowed to cool for hybridization. An illustration of thecomplementary oligonucleotides annealed to the specific oligomerscovalently immobilized at each location is shown in FIG. 15.

The hybridized array was then washed with a solution of 50 mM ammoniumcitrate buffer for cation exchange to remove sodium and potassium ionson the DNA backbone (Pieles, U. et al., (1993) Nucl. Acids Res.,21:3191-3196). A 6-nl aliquot of a matrix solution of 3-hydroxypicolinicacid [0.7 M 3-hydroxypicolinic acid-10% ammonium citrate in 50%acetonitrile; see Wu et al., Rapid Commun. Mass Spectrom. 7:142-146(1993)] was added to each location of the array using a piezoelectricpipette as described herein.

The solution was allowed to dry at ambient temperature and thereafter a6-nl aliquot of water was added to each location using a piezoelectricpipette to resuspend the dried matrix-DNA complex, such that upon dryingat ambient temperature the matrix-DNA complex forms a uniformcrystalline surface on the bottom surface of each location.

MALDI-TOF MS Analysis

The MALDI-TOF MS analysis was performed in series on each of the 16locations of the hybridization array illustrated in FIG. 15 essentiallyas described in EXAMPLE 1. The resulting mass spectrum ofoligonucleotides that specifically hybridized to each of the 16locations of the DNA hybridization array is shown in FIG. 16. The massspectrum revealed a specific signal at each location representative ofobserved experimental mass-to-charge ratio corresponding to the specificcomplementary nucleotide sequence.

For example, in the locations that have only Oligomer 1 conjugatedthereto, the mass spectrum revealed a predominate signal with anobserved experimental mass-to-charge ratio of 7072.4 approximately equalto that of the 23-mer; the theoretical mass-to-charge ratio of the23-mer is 7072.6 Da. Similarly, specific hybridization of the 12-meroligonucleotide to the array, observed experimental mass-to-charge ratioof 3618.33 Da (theoretical 3622.4 Da), was detected only at thoselocations conjugated with Oligomer 2 whereas specific hybridization ofMJM6 (observed experimental mass-to-charge ratio of 6415.4) was detectedonly at those locations of the array conjugated with Oligomer 3[theoretical 6407.2 Da].

None of the locations of the array revealed a signal that corresponds tothe negative control 29-mer oligonucleotide (theoretical mass-to-chargeratio of 8974.8) indicating that specific target DNA molecules can behybridized to oligomers covalently immobilized to specific locations onthe surface of the silicon array and a plurality of hybridization assaysmay be individually monitored using MALDI-TOF MS analysis.

(b) 8×8 (64-location) Array

A 2×2 cm² silicon wafer having 256 individual depressions or wells thatform a 16×16 array of wells was purchased from a commercial supplier[Accelerator Technology Corp., College Station, Tex.]. The wells were800×800 μm², 120 μm deep, on a 1.125 pitch. The silicon wafer wasreacted with 3-aminopropyltriethoxysilane to produce a uniform layer ofprimary amines on the surface and then exposed to the heterobifunctionalcrosslinker SIAB resulting in iodoacetamido functionalities on thesurface [e.g., see FIG. 7].

Following the procedures described above for the preparation of the16-location DNA array, Oligomers 1-3 were immobilized to 64 locationsforming an 8×8 array on the 256 well silicon wafer, hybridized tocomplementary oligonucleotides and analyzed by MALDI-TOF MS analysis.FIG. 17 shows the mass spectrum of the 64-location DNA array analyzed inseries by MALDI-TOF analysis. As shown for the 16-location array,specific hybridization of the complementary oligonucleotide to each ofthe immobilized thiol-containing oligomers was observed in each of thelocations of the DNA array.

EXAMPLE 6 Extension of Hybridized DNA Primers Bound to DNA TemplatesImmobilized on a Silicon Wafer

The SIAB-derivatized silicon wafers can also be employed for primerextension reactions of the immobilized DNA template using the proceduresessentially described in U.S. Pat. No. 5,605,798.

As shown in FIG. 18, a 27-mer oligonucleotide [SEQ ID NO: 11] containinga 3′-free thiol group was coupled to a SIAB-derivatized silicon wafer asdescribed above, for example, in Example 1. A 12-mer ligonucleotideprimer [SEQ ID NO: 12] was hybridized to the immobilized oligonucleotideand the primer was extended using a commercially available kit [e.g.,Sequenase or ThermoSequenase, U.S. Biochemical Corp]. The addition ofSequenase DNA polymerase or ThermoSequenase DNA polymerase in thepresence of three deoxyribonucleoside triphosphates (dNTPs; dATP, dGTP,dCTP) and dideoxyribonucleoside thymidine triphosphate (ddTTP) in bufferaccording to the instructions provided by the manufacturer resulted in a3-base extension of the 12-mer primer while still bound to the siliconwafer. The wafer was then analyzed by MALDI-TOF mass spectrometry asdescribed above. As shown in FIG. 18, the mass spectrum results clearlydistinguish the 15-mer [SEQ ID NO: 13] from the original unextended12-mer thus indicating that specific extension can be performed on thesurface of a silicon wafer and detected using MALDI-TOF MS analysis.

EXAMPLE 7

Effect of Linker Length on Polymerase Extension of Hybridized DNAPrimers Bound to DNA Templates Immobilized on a Silicon Wafer

The effect of the distance between the SIAB-conjugated silicon surfaceand the duplex DNA formed by hybridization of the target DNA to theimmobilized oligomer template was investigated, as well as choice ofenzyme [e.g., see FIG. 19].

Two SIAB-derivatized silicon wafers were conjugated to the 3′-end of twofree thiol-containing oligonucleotides of identical DNA sequence exceptfor a 3-base poly dT spacer sequence incorporated at the 3′-end [SEQ IDNOs: 8 & 11]. These two oligonuclotides were synthesized and each wasseparately immobilized to the surface of a silicon wafer through theSIAB cross-linker [e.g., see FIG. 7]. Each wafer was incubated with a12-mer oligonucleotide [SEQ ID NOs: 12, 14 and 15] complementary toportions of the nucleotide sequences common to both of theoligonucleotides by denaturing at 75° C. and slow cooling the siliconwafer. The wafers were then analyzed by MALDI-TOF mass spectrometry asdescribed above.

As previously shown in FIG. 18, a 3-base specific extension of the bound12-mer oligonucleotide was observed using the oligomer primer wherethere is a 9-base spacer between the duplex and the surface [SEQ ID NO:12]. As shown in FIG. 19, similar results were observed when the DNAspacer lengths between the SIAB moiety and the DNA duplex were 0, 3, 6and 12. The results of MALDI-TOF mass spectrometry analysis of thewafers are shown in FIG. 20. In addition, FIG. 19 also shows that theextension reaction may be performed using a variety of DNA polymerases.Thus, the SIAB linker may be directly coupled to the DNA template or mayinclude a linker sequence without effecting primer extension of thehybridized DNA.

EXAMPLE 8 Detection of Double-Stranded Nucleic Acid Molecules Via StrandDisplacement and Hybridization to an Immobilized Complementary NucleicAcid

This example describes immobilization of a 24-mer primer and thespecific hybridization of one strand of a duplex DNA molecule, therebypermitting amplification of a selected target molecule in solution phaseand permitting detection of the double stranded molecule.

A 24-mer DNA primer CTGATGCGTC GGATCATCTT TTTT [SEQ ID NO: 8],containing a 3′-free thiol group was coupled to a SIAB-derivatizedsilicon wafer essentially as outlined in FIG. 7 and described inExamples 1 and 2.

An 18-mer synthetic oligonucleotide 5′-CTGATGCGTCGGATCATC-3′ [SEQ ID NO:16] was premixed with a 12-mer oligonucleotide 5′-GATGATCCGACG-3′ [SEQID NO: 12] that has a sequence that is complementary to 12 base portionof the 18-mer oligonucleotide. The oligonucleotide mix was heated to 75°C. and cooled slowly to room temperature to facilitate the formation ofa duplex molecule: 5′-CTGATGCGTCGGATCATC-3′ [SEQ ID NO: 16]3′-GCAGCCTAGTAG-5′ [SEQ ID NO: 12].

The specific hybridization of the 12-mer strand of the duplex moleculeto the immobilized 24-mer primer was carried out by mixing 1 μM of theduplex molecule using the hybridization conditions described in Example6.

The wafers were analyzed by mass spectrometry as described above.Specific hybridization was detected in a mass spectrum of the 12-merwith a mass-to-charge ratio of 3682.78 Da.

EXAMPLE 91-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

A. 2-Nitro-5-(3-hydroxypropoxy)benzaldehyde

3-Bromo-1-propanol (3.34 g, 24 mmol) was refluxed in 80 ml of anhydrousacetonitrile with 5-hydroxy-2-nitrobenzaldehyde (3.34 g, 20 mmol), K₂CO₃(3.5 g), and Kl (100 mg) overnight (15 h). The reaction mixture wascooled to room temperature and 150 ml of methylene chloride was added.The mixture was filtered and the solid residue was washed with methylenechloride. The combined organic solution was evaporated to dryness andredissolved in 100 ml methylene chloride. The resulted solution waswashed with saturated NaCl solution and dried over sodium sulfate. 4.31g (96%) of desired product was obtained after removal of the solvent invacuo.

R_(f)=0.33 (dichloromethane/methanol, 95/5).

UV (methanol) maximum: 313, 240 (shoulder), 215 nm; minimum: 266 nm.

¹H NMR (DMSO-d₆) δ 10.28 (s, 1H), 8.17 (d, 1H), 7.35 (d, 1H), 7.22 (s,1H), 4.22(t, 2H), 3.54 (t, 2H), 1.90 (m, 2H).

¹³C NMR (DMSO-d₆) δ 189.9, 153.0, 141.6, 134.3, 127.3, 118.4, 114.0,66.2, 56.9, 31.7.

B. 2-Nitro-5-(3-O-t-butyidimethylsilylpropoxy)benzaldehyde

2-Nitro-5-(3-hydroxypropoxy)benzaldehyde(1 g, 4.44 mmol) was dissolvedin 50 ml anhydrous acetonitrile. To this solution, it was added 1 ml oftriethylamine, 200 mg of imidazole, and 0.8 g (5.3 mmol) of tBDMSCI. Themixture was stirred at room temperature for 4 h. Methanol (1 ml) wasadded to stop the reaction. The solvent was removed in vacuo and thesolid residue was redissolved in 100 ml methylene chloride. The resultedsolution was washed with saturated sodium bicarbonate solution and thenwater. The organic phase was dried over sodium sulfate and the solventwas removed in vacuo. The crude mixture was subjected to a quick silicagel column with methylene chloride to yield 1.44 g (96%) of2-nitro-5-(3-O-t-butyidimethylsilylpropoxy)benzaldehyde.

R_(f)=0.67 (hexane/ethyl acetate, 5/1).

UV (methanol), maximum: 317, 243, 215 nm; minimum: 235, 267 nm.

¹H NMR (DMSO-d₆) δ 10.28 (s, 1H), 8.14 (d, 1H), 7.32 (d, 1H), 7.20 (s,1H), 4.20 (t, 2H), 3.75 (t, 2H), 1.90 (m, 2H), 0.85 (s, 9H), 0.02 (s,6H).

¹³C NMR (DMSO-d₆) δ 189.6, 162.7, 141.5, 134.0, 127.1, 118.2, 113.8,65.4, 58.5, 31.2, 25.5, −3.1, −5.7.

C. 1-(2-Nitro-5-(3-O-t-butyidimethylsilylpropoxy)phenyl)ethanol

High vacuum dried2-nitro-5-(3-O-t-butyidimethylsilylpropoxy)benzaldehyde (1.02 g, 3 mmol)was dissolved 50 ml of anhydrous methylene chloride. 2 MTrimethylaluminium in toluene (3 ml) was added dropwise within 10 minand the reaction mixture was kept at room temperature. It was stirredfurther for 10 min and the mixture was poured into 10 ml ice cooledwater. The emulsion was separated from water phase and dried over 100 gof sodium sulfate to remove the remaining water. The solvent was removedin vacuo and the mixture was applied to a silica gel column withgradient methanol in methylene chloride. 0.94 g (86%) of desired productwas isolated.

R_(f)=0.375 (hexane/ethyl acetate, 5/1).

UV (methanol), maximum: 306, 233, 206 nm; minimum: 255, 220 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, ₁H), 7.36 (s, 1H), 7.00 (d, 1H), 5.49 (b,OH), 5.31 (q, 1H), 4.19 (m, 2H), 3.77 (t, 2H), 1.95 (m, 2H), 1.37 (d,3H), 0.86 (s, 9H), 0.04 (s, 6H).

¹³C NMR (DMSO-d₆) δ 162.6, 146.2, 139.6, 126.9, 112.9, 112.5, 64.8,63.9, 58.7, 31.5, 25.6, 24.9, −3.4, −5.8.

D. 1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol

1-(2-Nitro-5-(3-O-t-butyldimethylsilylpropoxy)phenyl)ethanol (0.89 g,2.5 mmol) was dissolved in 30 ml of THF and 0.5 mmol of nBU₄NF was addedunder stirring. The mixture was stirred at room temperature for 5 h andthe solvent was removed in vacuo. The remaining residue was applied to asilica gel column with gradient methanol in methylene chloride.1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol (0.6 g (99%) was obtained.

R_(f)=0.17 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 304, 232, 210 nm; minimum: 255, 219 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, 1H), 7.33 (s, 1H), 7.00 (d, 1H), 5.50 (d,OH), 5.28 (t, OH), 4.59 (t, 1H), 4.17 (t, 2H), 3.57 (m, 2H), 1.89 (m,2H), 1.36 (d, 2H).

¹³C NMR (DMOS-d₆) δ 162.8, 146.3, 139.7, 127.1, 113.1, 112.6, 65.5,64.0, 57.0, 31.8, 25.0.

E. 1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)ethanol

1-(2-Nitro-5-(3-hydroxypropoxy)phenyl)ethanol (0.482 g, 2 mmol) wasco-evaporated with anhydrous pyridine twice and dissolved in 20 mlanhydrous pyridine. The solution was cooled in ice-water bath and 750 mg(2.2 mmol) of DMTCI was added. The reaction mixture was stirred at roomtemperature overnight and 0.5 ml methanol was added to stop thereaction. The solvent was removed in vacuo and the residue wasco-evaporated with toluene twice to remove trace of pyridine. The finalresidue was applied to a silica gel column with gradient methanol inmethylene chloride containing drops of triethylamine to yield 0.96 g(89%) of the desired product1-(2-nitro-5-(3-O-4,4′-dimethoxytrityl-propoxy)phenyl)ethanol.

R_(f)=0.50 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 350 (shoulder), 305, 283, 276 (shoulder), 233,208 nm; minimum: 290, 258, 220 nm.

¹H NMR (DMSO-d₆) δ 8.00 (d, 1H), 6.82-7.42 (ArH), 5.52 (d, OH), 5.32 (m,1H), 4.23 (t, 2H), 3.71 (s, 6H), 3.17 (t, 2H), 2.00 (m, 2H), 1.37 (d,3H).

¹³C NMR (DMOS-d₆) δ 162.5, 157.9, 157.7, 146.1, 144.9, 140.1, 139.7,135.7, 129.5, 128.8, 127.6, 127.5, 127.3, 126.9, 126.4, 113.0, 112.8,112.6, 85.2, 65.3, 63.9, 59.0, 54.8, 28.9, 24.9.

F.1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

1-(2-Nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)ethanol (400 mg,0.74 mmol) was dried under high vacuum and was dissolved in 20 ml ofanhydrous methylene chloride. To this solution, it was added 0.5 mlN,N-diisopropylethylamine and 0.3 ml (1.34 mmol) of2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. The reaction mixturewas stirred at room temperature for 30 min and 0.5 ml of methanol wasadded to stop the reaction. The mixture was washed with saturated sodiumbicarbonate solution and was dried over sodium sulfate. The solvent wasremoved in vacuo and a quick silica gel column with 1% methanol inmethylene chloride containing drops of triethylamine yield 510 mg (93%)the desired phosphoramidite.

R_(f=)0.87 (dichloromethane/methanol, 99/1).

EXAMPLE 101-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

A. 4-(3-Hydroxypropoxy)-3-methoxyacetophenone

3-Bromo-1-propanol (53 ml, 33 mmol) was refluxed in 100 ml of anhydrousacetonitrile with 4-hydroxy-3-methoxyacetophenone (5 g, 30 mmol), K₂CO₃(5 g), and Kl (300 mg) overnight (15 h). Methylenechloride (150 ml) wasadded to the reaction mixture after cooling to room temperature. Themixture was filtered and the solid residue was washed with methylenechloride. The combined organic solution was evaporated to dryness andredissolved in 100 ml methylene chloride. The resulted solution waswashed with saturated NaCl solution and dried over sodium sulfate. 6.5 g(96.4%) of desired product was obtained after removal of the solvent invacuo.

R_(f)=0.41 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 304, 273, 227, 210 nm: minimum: 291, 244, 214nm.

¹H NMR (DMSO-d₆) δ 7.64 (d, 1H), 7.46 (s, 1H), 7.04 (d, 1H), 4.58 (b,OH), 4.12 (t, 2H), 3.80 (s, 3H), 3.56 (t, 2H), 2.54 (s, 3H), 1.88 (m,2H).

¹³C NMR (DMSO-d₆) δ 196.3, 152.5, 148.6, 129.7, 123.1, 111.5, 110.3,65.4, 57.2, 55.5, 31.9, 26.3.

B. 4-(3-Acetoxypropoxy)-3-methoxyacetophenone

4-(3-Hydroxypropoxy)-3-methoxyacetophenone (3.5 g, 15.6 mmol) was driedand dissolved in 80 ml anhydrous acetonitrile. This mixture, 6 ml oftriethylamine and 6 ml of acetic anhydride were added. After 4 h, 6 mlmethanol was added and the solvent was removed in vacuo. The residue wasdissolved in 100 ml dichloromethane and the solution was washed withdilute sodium bicarbonate solution, then water. The organic phase wasdried over sodium sulfate and the solvent was removed. The solid residuewas applied to a silica gel column with methylene chloride to yield 4.1g of 4-(3-acetoxypropoxy)-3-methoxyacetophenone (98.6%).

R_(f)=0.22 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 303, 273, 227, 210 nm; minimum: 290, 243, 214nm.

¹H NMR (DMSO-d₆) δ 7.62 (d, 1H), 7.45 (s, 1H), 7.08 (d, 1H), 4.12 (m,4H, 3.82 (s, 3H), 2.54 (s, 3H), 2.04 (m, 2H), 2.00 (s, 3H).

¹³C NMR (DMSO-d₆) δ 196.3, 170.4, 152.2, 148.6, 130.0, 123.0, 111.8,110.4, 65.2, 60.8, 55.5, 27.9, 26.3, 20.7.

C. 4-(3-Acetoxypropoxy)-3-methoxy-6-nitroacetophenone

4-(3-Acetoxypropoxy)-3-methoxyacetophenone (3.99 g, 15 mmol) was addedportionwise to 15 ml of 70% HNO₃ in water bath and keep the reactiontemperature at the room temperature. The reaction mixture was stirred atroom temperature for 30 min and 30 g of crushed ice was added. Thismixture was extracted with 100 ml of dichloromethane and the organicphase was washed with saturated sodium bicarbonate solution. Thesolution was dried over sodium sulfate and the solvent was removed invacuo. The crude mixture was applied to a silica gel column withgradient methanol in methylene chloride to yield 3.8 g (81.5%) ofdesired product 4-(3-acetoxypropoxy)-3-methoxy-6-nitroacetophenone and0.38 g (8%) of ipso-substituted product5-(3-acetoxypropoxy)-4-methoxy-1,2-dinitrobenzene. Side ipso-substitutedproduct 5-(3-acetoxypropoxy)-4-methoxy-1,2-dinitrobenzene:

R_(f)=0.47 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 334, 330, 270, 240, 212 nm; minimum: 310, 282,263, 223 nm.

¹H NMR (CDCl₃) δ 7.36 (s, 1H), 7.34 (s, 1H), 4.28 (t, 2H), 4.18 (t, 2H),4.02 (s, 3H), 2.20 (m, 2H), 2.08 (s, 3H).

¹³C NMR (CDCl³) δ 170.9, 152.2, 151.1, 117.6, 111.2, 107.9, 107.1, 66.7,60.6, 56.9, 28.2, 20.9.

Desired product 4-(3-acetoxypropoxy)-3-methoxy-6-nitroacetophenone:

R^(f=)0.29 (dichloromethane/methanol, 99/1).

UV (methanol), maximum: 344, 300, 246, 213 nm; minimum: 320, 270, 227nm.

¹H NMR (CDCl₃) δ 7.62 (s, 1H), 6.74 (s, 1H), 4.28 (t, 2H), 4.20 (t, 2H),3.96 (s, 3H), 2.48 (s, 3H), 2.20 (m, 2H), 2.08 (s, 3H).

¹³C NMR (CDCl₃) δ 200.0, 171.0, 154.3, 148.8, 138.3, 133.0, 108.8,108.0, 66.1, 60.8, 56.6, 30.4, 28.2, 20.9.

D. 1-(4-(3-Hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol

4-(3-Acetoxypropoxy)-3-methoxy-6-nitroacetophenone (3.73 g, 12 mmol) wasadded 150 ml ethanol and 6.5 g of K₂CO₃. The mixture was stirred at roomtemperature for 4 h and TLC with 5% methanol in dichloromethaneindicated the completion of the reaction. To this same reaction mixture,it was added 3.5 g of NaBH₄ and the mixture was stirred at roomtemperature for 2 h. Acetone (10 ml) was added to react with theremaining NaBH₄. The solvent was removed in vacuo and the residue wasuptaken into 50 g of silica gel. The silica gel mixture was applied onthe top of a silica gel column with 5% methanol in methylene chloride toyield 3.15 g (97%) of desired product1-(4-(3-hydroxypropoxy)-3-methoxy-6-nitrophenyl) ethanol. Intermediateproduct 4-(3-hydroxypropoxy)-3-methoxy-6-nitroacetophenone afterdeprotection:

R_(f)=0.60 (dichloromethane/methanol, 95/5).

Final product 1-(4-(3-hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol:

R_(f)=0.50 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 344, 300, 243, 219 nm: minimum: 317, 264, 233nm.

¹H NMR (DMSO-d₆) δ 7.54 (s, 1H), 7.36 (s, 1H), 5.47 (d, OH), 5.27 (m,1H), 4.55 (t, OH), 4.05 (t, 2H), 3.90 (s, 3H), 3.55 (q, 2H), 1.88 (m,2H), 1.37 (d, 3H).

¹³C NMR (DMSO-d₆) δ 153.4, 146.4, 138.8, 137.9, 109.0, 108.1, 68.5,65.9, 57.2, 56.0, 31.9, 29.6.

E.1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol

1-(4-(3-Hydroxypropoxy)-3-methoxy-6-nitrophenyl)ethanol (0.325 g, 1.2mmol) was co-evaporated with anhydrous pyridine twice and dissolved in15 ml anhydrous pyridine. The solution was cooled in ice-water bath and450 mg (1.33 mmol) of DMTCl was added. The reaction mixture was stirredat room temperature overnight and 0.5 ml methanol was added to stop thereaction. The solvent was removed in vacuo and the residue wasco-evaporated with toluene twice to remove trace of pyridine. The finalresidue was applied to a silica gel column with gradient methanol inmethylene chloride containing drops of triethylamine to yield 605 mg(88%) of desired product1-(4-(3-O-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol.

R_(f)=0.50 (dichloromethane/methanol, 95/5).

UV (methanol), maximum: 354, 302, 282, 274, 233, 209 nm; minimum: 322,292, 263, 222 nm.

¹H NMR (DMSO-d₆) δ 7.54 (s, 1H), 6.8-7.4 (ArH), 5.48 (d, OH), 5.27 (m,1H), 4.16 (t, 2H), 3.85 (s, 3H), 3.72 (s, 6H), 3.15 (t, 2H), 1.98 (t,2H), 1.37 (d, 3H).

¹³C NMR (DMSO-d₆) δ 157.8, 153.3, 146.1, 144.9, 138.7, 137.8, 135.7,129.4, 128.7, 127.5, 127.4, 126.3, 112.9, 112.6, 108.9, 108.2, 85.1,65.7, 63.7, 59.2, 55.8, 54.8, 29.0, 25.0.

F.1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane

1-(4-(3-O-4,4′-Dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)ethanol(200 mg, 3.5 mmol) was dried under high vacuum and was dissolved in 15ml of anhydrous methylene chloride. To this solution, it was added 0.5ml N,N-diisopropylethylamine and 0.2 ml (0.89 mmol) of2-cyaiioethyl-N,N-diisopropylchlorophosphoramidite. The reaction mixturewas stirred at room temperature for 30 min and 0.5 ml of methanol wasadded to stop the reaction. The mixture was washed with saturated sodiumbicarbonate solution and was dried over sodium sulfate. The solvent wasremoved in vacuo and a quick silica gel column with 1% methanol inmethylene chloride containing drops of triethylamine yield 247 mg(91.3%) the desired phosphoramidite1-(4-(3-O-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethane.

R_(f)=0.87 (dichloromethane/methanol, 99/1).

EXAMPLE 11 Oligonucleotide Synthesis

The oligonucleotide conjugates containing photocleavable linker wereprepared by solid phase nucleic acid synthesis (see: Sinha et al.Tetrahedron Lett. 1983, 24, 5843-5846; Sinha et al. Nucleic Acids Res.1984, 12, 4539-4557; Beaucage et al. Tetrahedron 1993, 49, 6123-6194;and Matteucci et al. J. Am. Chem. Soc. 1981, 103, 3185-3191) understandard conditions. In addition a longer coupling time period wasemployed for the incorporation of photocleavable unit and the 5′terminal amino group. The coupling efficiency was detected by measuringthe absorbance of released DMT cation and the results indicated acomparable coupling efficiency of phosphoramidite1-(2-nitro-5-(3-O-4,4′-dimethoxytritylpropoxy)phenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethaneor1-(4-(3-O-4,4′-dimethoxytritylpropoxy)-3-methoxy-6-nitrophenyl)-1-O-((2-cyanoethoxy)-diisopropylaminophosphino)ethanewith those of common nucleoside phosphoramodites. Deprotection of thebase protection and release of the conjugates from the solid support wascarried out with concentrated ammonium at 55° C. overnight. Deprotectionof the base protection of other conjugates was done by fast deprotectionwith AMA reagents. Purification of the MMT-on conjugates was done byHPLC (trityl-on) using 0.1 M triethylammonium acetate, pH 7.0 and agradient of acetonitrile (5% to 25% in 20 minutes). The collected MMT orDMT protected conjugate was reduced in volume, detritylated with 80%aqueous acetic acid (40 min, 0° C.), desalted, stored at −20° C.

EXAMPLE 12 Photolysis Study

In a typical case, 2 nmol of oligonucleotide conjugate containingphotocleavable linker in 200 μl distilled water was irradiated with along wavelength UV lamp (Blak Ray XX-15 UV lamp, Ultraviolet products,San Gabriel, Calif.) at a distance of 10 cm (emission peak 365 nm, lampintensity=1.1 mW/cm² at a distance of 31 cm). The resulting mixture wasanalyzed by HPLC (trityl-off) using 0.1 M triethylammonium acetate, pH7.0 and a gradient of acetonitrile. Analysis showed that the conjugatewas cleaved from the linder within minutes upon UV irradiation.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 19(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGAGC TCGGTACCCG G 21 (2)INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCGGGTACCG AGCTCGAATT C 21 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCTCTTGGGA ACTGTGTAGT ATT 23 (2)INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:112 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4: AGGCTGTCTC TCTCCCTCTC TCATACACACACACACACAC ACACACACAC ACACACACAC 60 ACACACACAC TCACACTCAC CCACANNNAAATACTACACA GTTCCCAAGA GG 112 (2) INFORMATION FOR SEQ ID NO: 5: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE:cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:<Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:TAATACGACT CACTATAGGG CGAAGGCTGT CTCTCTCCCT CTCTCATAC 49 (2) INFORMATIONFOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 135 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: TAATACGACT CACTATAGGG CGAAGGCTGT CTCTCTCCCTCTCTCATACA CACACACACA 60 CACACACACA CACACACACA CACACACACA CACTCACACTCACCCACANN NAAATACTAC 120 ACAGTTCCCA AGAGG 135 (2) INFORMATION FOR SEQID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 7: AATACTACAC AG 12 (2) INFORMATION FOR SEQ IDNO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 8: CTGATGCGTC GGATCATCTT TTTT 24 (2) INFORMATIONFOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 9: GATGATCCGA CGCATCAGAA TGT 23 (2) INFORMATIONFOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 10: GATCTAGCTG GGCCGAGCTA GGCCGTTGA 29 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTGATGCGTC GGATCATCTT TTTTTTT 27(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GATGATCCGA CG 12 (2)INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 13: GATGATCCGA CGCAT 15 (2) INFORMATIONFOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 14: AAAAAAGATG AT 12 (2) INFORMATION FOR SEQ IDNO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 15: GATCCGACGC AT 12 (2) INFORMATION FOR SEQ IDNO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii)MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 16: CTGATGCGTC GGATCATC 18 (2) INFORMATION FORSEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 17: GCCTGGTACA CTGCCAGGCG CTTCTGCAGG TCATCGGCATCGCGGAGGAG 50 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown>(vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:GCCTGGTACA CTGCCAGGCA CTTCTGCAGG TCATCGGCAT CGCGGAGGAG 50 (2)INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (v) FRAGMENT TYPE: <Unknown> (vi) ORIGINAL SOURCE: (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 19: GATGCCGATG ACCTGCAGAAG 21

What is claimed is:
 1. A method for synthesizing DNA on the surface of asupport, comprising: reacting the surface of a silicon support or asupport with a silicon surface with a solution of3-aminopropyltriethoxysilane to produce a uniform layer of primaryamines on the surface of the support; derivatizing the surface of thesupport with iodoacetamido functionalities by reacting the uniform layerof primary amines with a solution of. N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); contacting the surface of the support with athiol-containing strand of DNA, whereby the thiol-containing DNA isimmobilized on the surface of the support by a covalent bond between thethiol group of the thiol-containing DNA and the iodoacetamridofunctionality on the surface; hybridizing a single-strand of DNA that iscomplementary to a portion of the immobilized thiol-containing DNA; andadding at least one nucleotide to the 3′-end of the hybridizedsingle-strand of DNA by DNA synthesis, whereby DNA is synthesized on thesurface of a support; and determining a molecular weight by massspectrometry of the single-strand of DNA to which at least onenucleotide has been added.
 2. The method of claim 1, wherein theimmobilized DNA is positioned on the support in the form of an array. 3.The method of claim 1, further comprising: adding one or moredideoxynucleoside triphosphates during DNA synthesis.
 4. The method ofclaim 1, wherein the mass spectrometry analysis is selected from thegroup consisting of Matrix Assisted Laser Desorption/lonization,Time-of-Flight (MALDI-TOF) analysis, Electronspray Electrospray (ES),Ion Cyclotron Resonance (ICR) and Fourier transform.
 5. A method,comprising: reacting thiol-containing DNA molecules with a solid supportunder conditions such that a covalent bond is formed, therebyimmobilizing DNA molecules on the insoluble support, wherein: theresulting covalent linkages and support are stable to laser desorption,and the support is a silicon support or comprises a silicon surface or asilicon dioxide surface; hybridizing a single-strand of DNA to a portionof the immobilized thiol-containing DNA molecule complementary thereto;adding at least one deoxynucleotide or dideoxynucleotide to the 3′-endof the hybridized single strand of DNA by enzymatic DNA synthesis; anddetermining the molecular weight of the hybridized single-strand of DNAto which at least one deoxynucleotide or dideoxynucleotide has beenadded using mass spectrometry analysis, whereby the sequence of at leasta portion of thiol-containing DNA molecule immobilized on the surface ofa support is determined.
 6. A method for sequencing DNA, comprising:reacting the surface of a silicon support or support with a siliconsurface with a solution of 3-aminopropyltriethoxysilane to produce auniform layer of primary amines on the surface of the support,derivatizing the surface of the support with iodoacetamidofunctionalities by reacting the uniform layer of primary amines with asolution of N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), reactingthe surface of the support with a thiol-containing strand of DNA,whereby the thiol-containing DNA is immobilized on the surface of thesupport by a covalent bond between the thiol group of thethiol-containing DNA and the iodoacetamido functionality derivatized onthe surface of the support, hybridizing a single-strand of DNA to aportion of the immobilized thiol-containing DNA complementary thereto,performing DNA synthesis in the presence of an appropriate mixture ofdeoxynucleotides containing one or more dideoxynucleotides, wherein atleast one deoxynucleotide or dideoxynucleotide is added to thehybridized single-strand of DNA at its 3′-end by enzymatic DNAsynthesis; and determining the molecular weight of the hybridizedsingle-strand of DNA containing the enzymatically addeddeoxynucleotide(s) or dideoxynucleotide(s) using mass spectrometryanalysis, whereby at least one base in the sequence is determined. 7.The method of claim 5 or 6, wherein the mass spectrometry analysis isselected from the group consisting of Matrix Assisted LaserDesorption/lonization, Time-of-Flight (MALDI-TOF) analysis, Electrospray(ES), Ion Cyclotron Resonance (ICR) and Fourier transform.
 8. The methodof claim 5, or 6 wherein the immobilized DNA is positioned on thesupport in the form of an array.